Methods and organisms for converting synthesis gas or other gaseous carbon sources and methanol to 1,3-butanediol

ABSTRACT

A non-naturally occurring microbial organism having a 1,3-butanediol (1,3-BDO) pathway includes at least one exogenous nucleic acid encoding a 1,3-BDO pathway enzyme or protein expressed in a sufficient amount to produce 1,3-BDO. A method for producing 1,3-BDO that includes culturing the this non-naturally occurring microbial organism under conditions and for a sufficient period of time to produce 1,3-BDO.

This application claims the benefit of priority of U.S. provisionalapplication Ser. No. 61/285,312, filed Dec. 10, 2009, the entirecontents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to biosynthetic processes andmore specifically to organisms capable of using carbohydrates, methanol,synthesis gas and other gaseous carbon sources in the production ofcommodity chemicals.

1,3-butanediol (1,3-BDO) is a four carbon diol traditionally producedfrom acetylene via its hydration. The resulting acetaldehyde is thenconverted to 3-hydroxybutyraldehdye which is subsequently reduced toform 1,3-BDO. In more recent years, acetylene has been replaced by theless expensive ethylene as a source of acetaldehyde. 1,3-BDO is commonlyused as an organic solvent for food flavoring agents. It is also used asa co-monomer for polyurethane and polyester resins and is widelyemployed as a hypoglycaemic agent. Optically active 1,3-BDO is a usefulstarting material for the synthesis of biologically active compounds andliquid crystals. Another use of 1,3-butanediol is that its dehydrationaffords 1,3-butadiene (Ichikawa et al., J. Molecular CatalysisA-Chemical, 231:181-189 (2005); Ichikawa et al., J. Molecular CatalysisA-Chemical, 256:106-112 (2006)), a chemical used to manufacturesynthetic rubbers (e.g. tires), latex, and resins.

Synthesis gas (syngas) is a mixture of primarily H₂ and CO that can beobtained via gasification of any organic feedstock, such as coal, coaloil, natural gas, biomass, or waste organic matter. Numerousgasification processes have been developed, and most designs are basedon partial oxidation, where limiting oxygen avoids full combustion, oforganic materials at high temperatures (500-1500° C.) to provide syngasas a 0.5:1-3:1 H₂/CO mixture. Steam is sometimes added to increase thehydrogen content, typically with increased CO₂ production through thewater gas shift reaction. Methanol is most commonly producedindustrially from the syngas components, CO, and H₂, via catalysis.

Today, coal is the main substrate used for industrial production ofsyngas, which is traditionally used for heating and power and as afeedstock for Fischer-Tropsch synthesis of methanol and liquidhydrocarbons. Many large chemical and energy companies employ coalgasification processes on large scale and there is experience in theindustry using this technology.

Moreover, technology now exists for cost-effective production of syngasfrom a plethora of other materials such as biomass, wastes, polymers,and the like, at virtually any location in the world. The benefits ofusing syngas include flexibility, since syngas can be produced from mostorganic substances, including biomass. Another benefit is that syngas isinexpensive. In addition, there are known pathways, as in organisms suchas Clostridium spp., that utilize syngas effectively.

Despite the availability of organisms that utilize syngas, in generalthe known organisms are poorly characterized and are not well suited forcommercial development. For example, Clostridium and related bacteriaare strict anaerobes that are intolerant to high concentrations ofcertain products such as butanol, thus limiting titers andcommercialization potential. The Clostridia also produce multipleproducts, which presents separations issues in obtaining a desiredproduct. Finally development of facile genetic tools to manipulateClostridial genes is in its infancy; therefore, they are not readilyamenable to genetic engineering to improve yield or productioncharacteristics of a desired product.

Increasing the flexibility of inexpensive and readily availablefeedstocks while minimizing the environmental impact of chemicalproduction are two goals of a sustainable chemical industry. Feedstockflexibility relies on the introduction of methods that enable access anduse of a wide range of materials as primary feedstocks for chemicalmanufacturing. The reliance on petroleum based feedstocks for eitheracetylene or ethylene warrants the development of a renewable feedstockbased route to 1,3-butanediol and to butadiene.

Thus, there exists a need to develop microorganisms and methods of theiruse to utilize carbohydrates, methanol, syngas or other gaseous carbonsources for the production of 1,3-butanediol. The present inventionsatisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

In some aspects, embodiments disclosed herein relate to a non-naturallyoccurring microbial organism having a 1,3-butanediol (1,3-BDO) pathwaythat includes at least one exogenous nucleic acid encoding a 1,3-BDOpathway enzyme or protein expressed in a sufficient amount to produce1,3-BDO. The 1,3-BDO pathway includes Methanol methyltransferase (MtaB),Corrinoid protein (MtaC), Methyltetrahydrofolate:corrinoid proteinmethyltransferase (MtaA), Methyltetrahydrofolate:corrinoid proteinmethyltransferase (AcsE), Corrinoid iron-sulfur protein (AcsD),Nickel-protein assembly protein (AcsF & CooC), Ferredoxin (Orf7),Acetyl-CoA synthase (AcsB & AcsC), Carbon monoxide dehydrogenase (AcsA),Hydrogenase (Hyd), Acetoacetyl-CoA thiolase (AtoB), Acetoacetyl-CoAreductase (CoA-dependent, aldehyde forming), 3-oxobutyraldehydereductase (ketone reducing), 3-hydroxybutyraldehyde reductase,Acetoacetyl-CoA reductase (CoA-dependent, alcohol forming),3-oxobutyraldehyde reductase (aldehyde reducing), 4-hydroxy,2-butanonereductase, Acetoacetyl-CoA reductase (ketone reducing),3-hydroxybutyryl-CoA reductase (aldehyde forming), 3-hydroxybutyryl-CoAreductase (alcohol forming), 3-hydroxybutyryl-CoA transferase,3-hydroxybutyryl-CoA hydrolase, 3-hydroxybutyryl-CoA synthetase,3-hydroxybutyrate dehydrogenase, 3-hydroxybutyrate reductase,Acetoacetyl-CoA transferase, Acetoacetyl-CoA hydrolase, Acetoacetyl-CoAsynthetase, or Acetoacetate reductase.

In some aspects, embodiments disclosed herein relate to a non-naturallyoccurring microbial organism having a 1,3-butanediol (1,3-BDO) pathwaythat includes at least one exogenous nucleic acid encoding a 1,3-BDOpathway enzyme or protein expressed in a sufficient amount to produce1,3-BDO. The 1,3-BDO pathway includes Formate dehydrogenase,Formyltetrahydrofolate synthetase, Methenyltetrahydrofolatecyclohydrolase, Methylenetetrahydrofolate dehydrogenase,Methylenetetrahydrofolate reductase, Methyltetrahydrofolate:corrinoidprotein methyltransferase (AcsE), Corrinoid iron-sulfur protein (AcsD),Nickel-protein assembly protein (AcsF & CooC), Ferredoxin (Orf7),Acetyl-CoA synthase (AcsB & AcsC), Carbon monoxide dehydrogenase (AcsA),Hydrogenase (Hyd), Acetoacetyl-CoA thiolase (AtoB), Acetoacetyl-CoAreductase (CoA-dependent, aldehyde forming), 3-oxobutyraldehydereductase (ketone reducing), 3-hydroxybutyraldehyde reductase,Acetoacetyl-CoA reductase (CoA-dependent, alcohol forming),3-oxobutyraldehyde reductase (aldehyde reducing), 4-hydroxy,2-butanonereductase, Acetoacetyl-CoA reductase (ketone reducing),3-hydroxybutyryl-CoA reductase (aldehyde forming), 3-hydroxybutyryl-CoAreductase (alcohol forming), 3-hydroxybutyryl-CoA transferase,3-hydroxybutyryl-CoA hydrolase, 3-hydroxybutyryl-CoA synthetase,3-hydroxybutyrate dehydrogenase, 3-hydroxybutyrate reductase,Acetoacetyl-CoA transferase, Acetoacetyl-CoA hydrolase, Acetoacetyl-CoAsynthetase, or Acetoacetate reductase.

In some aspects, embodiments disclosed herein relate to a method forproducing 1,3-BDO that includes culturing the aforementionednon-naturally occurring microbial organisms under conditions and for asufficient period of time to produce 1,3-BDO.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram depicting the Wood-Ljungdahl pathway andformation routes for acetate and ethanol. The transformations that arecharacteristic of organisms capable of growth on synthesis gas are 1) COdehydrogenase, 2) hydrogenase, 3) energy-conserving hydrogenase (ECH),and 4) bi-functional CO dehydrogenase/acetyl-CoA synthase.

FIG. 2 shows the complete Wood-Ljungdahl pathway that enables theconversion of gases including CO, CO₂, and/or H₂ to acetyl-CoA which issubsequently converted to cell mass and products such as ethanol oracetate. Abbreviations: 10FTHF: 10-formyltetrahydrofolate, 5MTHF:5-methyltetrahydrofolate, ACTP: acetyl phosphate, CFeSp: corrinoid ironsulfur protein, FOR: formate, MeOH: methanol, METHF:methyltetrahydrofolate, MLTHF: metheneyltetrahydrofolate, THF:tetrahydrofolate.

FIG. 3 depicts pathways from acetoacetyl-CoA to 1,3-butanediol. Theenzymatic steps are: A) acetoacetyl-CoA reductase (CoA-dependent,aldehyde forming), B) 3-oxobutyraldehyde reductase (ketone reducing), C)3-hydroxybutyraldehyde reductase, D) acetoacetyl-CoA reductase(CoA-dependent, alcohol forming), E) 3-oxobutyraldehyde reductase(aldehyde reducing), F) 4-hydroxy,2-butanone reductase, G)acetoacetyl-CoA reductase (ketone reducing), H) 3-hydroxybutyryl-CoAreductase (aldehyde forming), I) 3-hydroxybutyryl-CoA reductase (alcoholforming), J) 3-hydroxybutyryl-CoA transferase, hydrolase, or synthetase,K) 3-hydroxybutyrate dehydrogenase, L) 3-hydroxybutyrate reductase, M)acetoacetyl-CoA transferase, hydrolase, or synthetase N) acetoacetatereductase.

FIG. 4 shows a biosynthetic metabolic pathway for the conversion ofcarbohydrates, such as glucose, gases including CO, CO₂, and/or H₂, andmethanol to acetyl-CoA and further to 1,3-butanediol. The specificenzymatic transformations that are engineered into a production host arenumbered and shown in the figure. Abbreviations: 10FTHF:10-formyltetrahydrofolate, 5MTHF: 5-methyltetrahydrofolate, ACTP: acetylphosphate, CFeSp: corrinoid iron sulfur protein, FOR: formate, MeOH:methanol, METHF: methyltetrahydrofolate, MLTHF:metheneyltetrahydrofolate, THF: tetrahydrofolate.

FIG. 5 shows a biosynthetic metabolic pathway for the conversion ofcarbohydrates, such as glucose, gases including CO, CO₂, and/or H₂ andmethanol to acetyl-CoA, and further to 1,3-butanediol. The specificenzymatic transformations that are engineered into a production host arenumbered and shown in the figure. Abbreviations: 10FTHF:10-formyltetrahydrofolate, 5MTHF: 5-methyltetrahydrofolate, ACTP: acetylphosphate, CFeSp: corrinoid iron sulfur protein, FOR: formate, MeOH:methanol, METHF: methyltetrahydrofolate, MLTHF:metheneyltetrahydrofolate, THF: tetrahydrofolate.

FIG. 6 shows Western blots of 10 micrograms ACS90 (lane 1), ACS91(lane2), Mta98/99 (lanes 3 and 4) cell extracts with size standards(lane 5) and controls of M. thermoacetica CODH (Moth_(—)1202/1203) orMtr (Moth_(—)1197) proteins (50, 150, 250, 350, 450, 500, 750, 900, and1000 ng).

FIG. 7 shows cuvettes used in a methyl viologen assay. A blank is on theright and a cuvette with reduced methyl viologen is on the left. Note,the stoppers and vacuum grease on top of each are used to keep thereactions anaerobic.

FIG. 8 shows a spectrogram of ACS90 cell extracts assayed for transferof CH₃ from added CH₃-THF to purified M. thermoacetica corrinoidprotein.

FIG. 9 shows from left to right, anaerobic growth of recombinant E. coliMG1655 in N₂ and CO for 36 hr at 37° C., empty vector, ACS90, and ACS91.

FIG. 10 shows an exemplary flux distribution that can produce a 1.09mol/mol yield of 1,3-butanediol on glucose by emplying theWood-Ljungdahl pathway enzymes in combination with a 1,3-butanediolbiosynthetic pathway.

FIG. 11 shows an exemplary flux distribution that can produce a 1.2mol/mol yield of 1,3-butanediol on glucose by employing enzymes thatallow the co-utilization of methanol as a substrate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to developing and using microorganismscapable of utilizing carbohydrates, methanol, syngas and/or othergaseous carbon sources to produce 1,3-butanediol. The invention furtherrelates to expanding the product range of syngas-utilizingmicroorganisms and generating recombinant organisms capable of utilizingsyngas to produce 1,3-butanediol and optimizing their yields, titers,and productivities. Development of a recombinant organism, for example,Escherichia coli or other organisms suitable for commercial scale up,that can efficiently utilize syngas as a substrate for growth and forchemical production provides cost-advantaged processes for renewablechemical and fuel manufacturing. The organisms can be optimized andtested rapidly and at reasonable costs.

The potential of syngas as a feedstock resides in its ability to beefficiently and cost-effectively converted into chemicals and fuels ofinterest. Two main technologies for syngas conversion areFischer-Tropsch processes and fermentative processes. TheFischer-Tropsch (F-T) technology has been developed since World War IIand involves inorganic and metal-based catalysts that allow efficientproduction of methanol or mixed hydrocarbons as fuels. The drawbacks ofF-T processes are: 1) a lack of product selectivity, which results indifficulties separating desired products; 2) catalyst sensitivity topoisoning; 3) high energy costs due to high temperatures and pressuresrequired; and 4) the limited range of products available at commerciallycompetitive costs.

For fermentative processes, syngas has been shown to serve as a carbonand energy source for many anaerobic microorganisms that can convertthis material into products such as ethanol, acetate and hydrogen. Themain benefits of fermentative conversion of syngas are the selectivityof organisms for production of single products, greater tolerance tosyngas impurities, lower operating temperatures and pressures, andpotential for a large portfolio of products from syngas. The maindrawbacks of fermentative processes are that organisms known to convertsyngas tend to generate only a limited range of chemicals, such asethanol and acetate, and are not efficient producers of other chemicals,the organisms lack established tools for genetic manipulation, and theorganisms are sensitive to end products at high concentrations.

The present invention relates to the generation of microorganisms thatare effective at producing 1,3-butanediol from syngas or other gaseouscarbon sources. The organisms and methods of the present invention allowproduction of 1,3-butanediol at costs that are significantly advantagedover both traditional petroleum-based products and products deriveddirectly from glucose, sucrose or lignocellulosic sugars. In oneembodiment, the invention provides a non-naturally occurringmicroorganism capable of utilizing syngas or other gaseous carbonsources to produce 1,3-butanediol in which the parent microorganismlacks the natural ability to utilize syngas, as shown in FIGS. 4 and 5.In such microorganisms, one or more proteins or enzymes are expressed inthe microorganism, thereby conferring a pathway to utilize syngas orother gaseous carbon source to produce a desired product. In otherembodiments, the invention provides a non-naturally occurringmicroorganism that has been genetically modified, for example, byexpressing one or more exogenous proteins or enzymes that confer anincreased efficiency of production of 1,3-butanediol, where the parentmicroorganism has the ability to utilize syngas or other gaseous carbonsource to produce a desired product. Thus, the invention relates togenerating a microorganism with a new metabolic pathway capable ofutilizing syngas as well as generating a microorganism with improvedefficiency of utilizing syngas or other gaseous carbon source to produce1,3-butanediol.

The present invention additionally provides a non-naturally occurringmicroorganism expressing genes encoding enzymes that catalyze reactionsassociated with the carbonyl-branch of the Wood-Ljungdahl pathway inconjunction with a MtaABC-type methyltransferase system. Such anorganism is capable of converting methanol, a relatively inexpensiveorganic feedstock that can be derived from synthesis gas, and gasescomprising CO, CO₂, and/or H₂ into acetyl-CoA, cell mass, and products.The present invention further provides pathways that can achieve anincreased yield of 1,3-butandiol on carbohydrate feedstocks over whatwould be naturally expected, that is about 1 mol 1,3-butanediol/mol ofglucose, by providing an efficient mechanism for fixing the carbonpresent in methanol or carbon dioxide, fed exogenously or producedendogenously, into acetyl-CoA.

Escherichia coli is an industrial workhorse organism with an unrivaledsuite of genetic tools. Engineering the capability to convert CO₂, Co,and/or H₂ into acetyl-CoA, the central metabolite from which all cellmass components and many valuable products can be derived, into aforeign host such as E. coli can be accomplished following theexpression of exogenous genes that encode various proteins of theWood-Ljungdahl pathway. This pathway is highly active in acetogenicorganisms such as Moorella thermoacetica (formerly, Clostridiumthermoaceticum), which has been the model organism for elucidating theWood-Ljungdahl pathway since its isolation in 1942 (Fontaine et al., JBacteriol. 43:701-715 (1942)). The Wood-Ljungdahl pathway comprises twobranches: the Eastern, or methyl, branch that allows the conversion ofCO₂ to methyltetrahydrofolate (Me-THF) and the Western, or carbonyl,branch that allows the conversion of methyl-THF, CO, and Coenzyme-A intoacetyl-CoA (see FIGS. 1 and 2). As disclosed herein, the inventionprovides a non-naturally occurring microorganism expressing genes thatcatalyze both branches of the Wood-Ljungdahl pathway, in addition togenes for the production of 1,3-butanediol. Such an organism is capableof converting gases comprising CO, CO2, and/or H₂ into acetyl-CoA,1,3-butanediol, cell mass, and products. Such an organism is alsocapable of producing 1,3-butanediol from carbohydrates at thestoichiometric optimum yield. For example, in combination with any ofthe acetyl-CoA to 1,3-butanediol pathways, the Wood-Ljungdahl enzymesprovide the means to produce 12 moles of 1,3-butanediol for every 11moles of glucose as opposed to 1 mol 1,3-butanediol/1 mol glucose whichwould be attainable in the absence of the Wood-Ljungdahl pathwayenzymes.

The invention additionally provides a non-naturally occurringmicroorganism expressing genes encoding enzymes that catalyze thecarbonyl-branch of the Wood-Ljungdahl pathway in conjunction with aMtaABC-type methyltransferase system, in addition to genes for theproduction of 1,3-butanediol. Such an organism is capable of convertingmethanol, a relatively inexpensive organic feedstock that can be derivedfrom synthesis gas, and gases comprising CO, CO₂, and/or H₂ intoacetyl-CoA, 1,3-butanediol, cell mass, and products. In addition togaseous substrates, the organism can utilize methanol exclusively or incombination with carbohydrate feedstocks such as glucose to produce1,3-butanediol at high yield.

Synthesis gas, also known as syngas or producer gas, is the majorproduct of gasification of coal and of carbonaceous materials such asbiomass materials, including agricultural crops and residues. Syngas isa mixture primarily of H₂ and CO and can be obtained from thegasification of any organic feedstock, including but not limited tocoal, coal oil, natural gas, biomass, and waste organic matter.Gasification is generally carried out under a high fuel to oxygen ratio.Although largely H₂ and CO, syngas can also include CO₂ and other gasesin smaller quantities. Thus, synthesis gas provides a cost effectivesource of gaseous carbon such as CO and, additionally, CO₂.

As disclosed herein, gaseous carbon sources such as syngas comprising COand/or CO₂ can be utilized by non-naturally occurring microorganisms ofthe invention to produce 1,3-butanediol. Although generally exemplifiedherein as syngas, it is understood that any source of gaseous carboncomprising CO and/or CO₂ can be utilized by the non-naturally occurringmicroorganisms of the invention. Thus, the invention relates tonon-naturally occurring microorganisms that are capable of utilizing COand/or CO₂ as a carbon source.

The Wood-Ljungdahl pathway catalyzes the conversion of CO and H₂ toacetyl-CoA and other products such as acetate. Organisms capable ofutilizing CO and syngas also generally have the capability of utilizingCO₂ and CO₂/H₂ mixtures through the same basic set of enzymes andtransformations encompassed by the Wood-Ljungdahl pathway. H₂-dependentconversion of CO₂ to acetate by microorganisms was recognized longbefore it was revealed that CO also could be used by the same organismsand that the same pathways were involved. Many acetogens have been shownto grow in the presence of CO₂ and produce compounds such as acetate aslong as hydrogen is present to supply the necessary reducing equivalents(see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, NewYork, (1994)). This can be summarized by the following equation:2CO₂+4H₂ +nADP+nPi→CH₃COOH+2H₂O+nATPHence, non-naturally occurring microorganisms possessing theWood-Ljungdahl pathway can utilize CO₂ and H₂ mixtures as well for theproduction of acetyl-CoA and other desired products, such as1,3-butanediol.

The Wood-Ljungdahl pathway is well known in the art and consists of 12reactions which can be separated into two branches: (1) methyl branchand (2) carbonyl branch. The methyl branch converts syngas tomethyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branchconverts methyl-THF to acetyl-CoA. The reactions in the methyl branchare catalyzed in order by the following enzymes or proteins: ferredoxinoxidoreductase, formate dehydrogenase, formyltetrahydrofolatesynthetase, methenyltetrahydrofolate cyclodehydratase,methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolatereductase. The reactions in the carbonyl branch are catalyzed in orderby the following enzymes or proteins: methyltetrahydrofolate:corrinoidprotein methyltransferase (for example, AcsE), corrinoid iron-sulfurprotein, nickel-protein assembly protein (for example, AcsF),ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase andnickel-protein assembly protein (for example, CooC). Following theteachings and guidance provided herein for introducing a sufficientnumber of encoding nucleic acids to generate an acetyl-CoA pathway,those skilled in the art will understand that the same engineeringdesign also can be performed with respect to introducing at least thenucleic acids encoding the Wood-Ljungdahl enzymes or proteins absent inthe host organism. Therefore, introduction of one or more encodingnucleic acids into the microbial organisms of the invention such thatthe modified organism contains one branch or the complete Wood-Ljungdahlpathway will confer syngas utilization ability.

Thus, the non-naturally occurring microorganisms of the invention canuse syngas or other gaseous carbon sources providing CO and/or CO₂ toproduce 1,3-butanediol. In the case of CO₂, additional sources include,but are not limited to, production of CO₂ as a byproduct in ammonia andhydrogen plants, where methane is converted to CO₂; combustion of woodand fossil fuels; production of CO₂ as a byproduct of fermentation ofsugar in the brewing of beer, whisky and other alcoholic beverages, orother fermentative processes; thermal decomposition of limestone, CaCO₃,in the manufacture of lime, CaO; production of CO₂ as byproduct ofsodium phosphate manufacture; and directly from natural carbon dioxidesprings, where it is produced by the action of acidified water onlimestone or dolomite.

Acetogens, such as Moorella thermoacetica, C. ljungdahlii and C.carboxidivorans, can grow on a number of carbon sources ranging fromhexose sugars to carbon monoxide. Hexoses, such as glucose, aremetabolized first via Embden-Meyerhof-Parnas (EMP) glycolysis topyruvate, which is then converted to acetyl-CoA via pyruvate:ferredoxinoxidoreductase (PFOR). Acetyl-CoA can be used to build biomassprecursors or can be converted to acetate which produces energy viaacetate kinase and phosphotransacetylase. The overall conversion ofglucose to acetate, energy, and reducing equivalents isC₆H₁₂O₆+4ADP+4Pi→2CH₃COOH+2CO₂+4ATP+8[H]

Acetogens extract even more energy out of the glucose to acetateconversion while also maintaining redox balance by further convertingthe released CO₂ to acetate via the Wood-Ljungdahl pathway:2CO₂+8[H]+nADP+nPi→CH₃COOH+nATP

The coefficient n in the above equation signifies that this conversionis an energy generating endeavor, as many acetogens can grow in thepresence of CO₂ via the Wood-Ljungdahl pathway even in the absence ofglucose as long as hydrogen is present to supply the necessary reducingequivalents.2CO₂+4H₂ +nADP+nPi→CH₃COOH+2H₂O+nATP

The Wood-Ljungdahl pathway, illustrated in FIG. 1, is coupled to thecreation of Na⁺ or H⁺ ion gradients that can generate ATP via an Na⁺- orH⁺-dependant ATP synthase, respectively Muller V. Energy conservation inacetogenic bacteria. Appl Environ Microbiol 69:6345-6353 (2003). Basedon these known transformations, acetogens also have the capacity toutilize CO as the sole carbon and energy source. Specifically, CO can beoxidized to produce reducing equivalents and CO₂, or directlyassimilated into acetyl-CoA which is subsequently converted to eitherbiomass or acetate.4CO+2H₂O→CH₃COOH+2CO₂

Even higher acetate yields, however, can be attained when enoughhydrogen is present to satisfy the requirement for reducing equivalents.2CO+2H₂→CH₃COOH

Following from FIG. 1, the production of acetate via acetyl-CoAgenerates one ATP molecule, whereas the production of ethanol fromacetyl-CoA does not and requires two reducing equivalents. Thus, it canbe concluded that ethanol production from syngas will not generatesufficient energy for cell growth in the absence of acetate production.However, under certain conditions, Clostridium ljungdahlii producesmostly ethanol from synthesis gas (Klasson et al., Fuel 72.12:1673-1678(1993)) indicating that some combination of the pathways does indeedgenerate enough energy to support cell growth.2CO₂+6H₂→CH₃CH₂OH+3H₂O6CO+3H₂O→CH₃CH₂OH+4CO₂2CO+4H₂→CH₃CH₂OH+H₂O

Hydrogenic bacteria such as R. rubrum can also generate energy from theconversion of CO and water to hydrogen (see FIG. 1) (Simpma et al.,Critical Reviews in Biotechnology, 26.1:41-65 (2006)). A centralmechanism is the coordinated action of an energy converting hydrogenase(ECH) and CO dehydrogenase. The CO dehydrogenase supplies electrons fromCO which are then used to reduce protons to H₂ by ECH, whose activity iscoupled to energy-generating proton translocation. The net result is thegeneration of energy via the water-gas shift reaction.

The processes disclosed herein for the biosynthetic production of1,3-BDO via syngas involve sustainable manufacturing practices thatutilize renewable feedstocks, reduce energy intensity and lowergreenhouse gas emissions. Moreover, the dehydration of biobased-1,3-BDOrepresents a renewable route to butadiene in small end-use facilitieswhere no transport of this flammable and reactive chemical is required.

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism of the invention isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial organism's genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. Exemplarymetabolic polypeptides include enzymes or proteins within a1,3-butanediol biosynthetic pathway.

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, non-naturallyoccurring microorganisms can have genetic modifications to nucleic acidsencoding metabolic polypeptides or, functional fragments thereof.Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to amicrobial organism is intended to mean an organism that is substantiallyfree of at least one component as the referenced microbial organism isfound in nature. The term includes a microbial organism that is removedfrom some or all components as it is found in its natural environment.The term also includes a microbial organism that is removed from some orall components as the microbial organism is found in non-naturallyoccurring environments. Therefore, an isolated microbial organism ispartly or completely separated from other substances as it is found innature or as it is grown, stored or subsisted in non-naturally occurringenvironments. Specific examples of isolated microbial organisms includepartially pure microbes, substantially pure microbes and microbescultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or“microorganism” is intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

As used herein, the term “CoA” or “coenzyme A” is intended to mean anorganic cofactor or prosthetic group (nonprotein portion of an enzyme)whose presence is required for the activity of many enzymes (theapoenzyme) to form an active enzyme system. Coenzyme A functions incertain condensing enzymes, acts in acetyl or other acyl group transferand in fatty acid synthesis and oxidation, pyruvate oxidation and inother acetylation.

As used herein, the term “substantially anaerobic” when used inreference to a culture or growth condition is intended to mean that theamount of oxygen is less than about 10% of saturation for dissolvedoxygen in liquid media. The term also is intended to include sealedchambers of liquid or solid medium maintained with an atmosphere of lessthan about 1% oxygen.

“Exogenous” as it is used herein is intended to mean that the referencedmolecule or the referenced activity is introduced into the hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid of theinvention can utilize either or both a heterologous or homologousencoding nucleic acid.

It is understood that when more than one exogenous nucleic acid isincluded in a microbial organism that the more than one exogenousnucleic acid refers to the referenced encoding nucleic acid orbiosynthetic activity, as discussed above. It is further understood, asdisclosed herein, that such more than one exogenous nucleic acids can beintroduced into the host microbial organism on separate nucleic acidmolecules, on polycistronic nucleic acid molecules, or a combinationthereof, and still be considered as more than one exogenous nucleicacid. For example, as disclosed herein a microbial organism can beengineered to express two or more exogenous nucleic acids encoding adesired pathway enzyme or protein. In the case where two exogenousnucleic acids encoding a desired activity are introduced into a hostmicrobial organism, it is understood that the two exogenous nucleicacids can be introduced as a single nucleic acid, for example, on asingle plasmid, on separate plasmids, can be integrated into the hostchromosome at a single site or multiple sites, and still be consideredas two exogenous nucleic acids. Similarly, it is understood that morethan two exogenous nucleic acids can be introduced into a host organismin any desired combination, for example, on a single plasmid, onseparate plasmids, can be integrated into the host chromosome at asingle site or multiple sites, and still be considered as two or moreexogenous nucleic acids, for example three exogenous nucleic acids.Thus, the number of referenced exogenous nucleic acids or biosyntheticactivities refers to the number of encoding nucleic acids or the numberof biosynthetic activities, not the number of separate nucleic acidsintroduced into the host organism.

The non-naturally occurring microbal organisms of the invention cancontain stable genetic alterations, which refers to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications can be greater than 50generations, including indefinitely.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein, are described withreference to a suitable host organism such as E. coli and theircorresponding metabolic reactions or a suitable source organism fordesired genetic material such as genes for a desired metabolic pathway.However, given the complete genome sequencing of a wide variety oforganisms and the high level of skill in the area of genomics, thoseskilled in the art will readily be able to apply the teachings andguidance provided herein to essentially all other organisms. Forexample, the E. coli metabolic alterations exemplified herein canreadily be applied to other species by incorporating the same oranalogous encoding nucleic acid from species other than the referencedspecies. Such genetic alterations include, for example, geneticalterations of species homologs, in general, and in particular,orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less that 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities. Members of theserine protease family of enzymes, including tissue plasminogenactivator and elastase, are considered to have arisen by verticaldescent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the production of abiochemical product, those skilled in the art will understand that theorthologous gene harboring the metabolic activity to be introduced ordisrupted is to be chosen for construction of the non-naturallyoccurring microorganism. An example of orthologs exhibiting separableactivities is where distinct activities have been separated intodistinct gene products between two or more species or within a singlespecies. A specific example is the separation of elastase proteolysisand plasminogen proteolysis, two types of serine protease activity, intodistinct molecules as plasminogen activator and elastase. A secondexample is the separation of mycoplasma 5′-3′ exonuclease and DrosophilaDNA polymerase III activity. The DNA polymerase from the first speciescan be considered an ortholog to either or both of the exonuclease orthe polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor. Groups of paralogousprotein families include HipA homologs, luciferase genes, peptidases,and others.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement can beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene productcompared to a gene encoding the function sought to be substituted.Therefore, a nonorthologous gene includes, for example, a paralog or anunrelated gene.

Therefore, in identifying and constructing the non-naturally occurringmicrobial organisms of the invention having 1,3-butanediol biosyntheticcapability, those skilled in the art will understand with applying theteaching and guidance provided herein to a particular species that theidentification of metabolic modifications can include identification andinclusion or inactivation of orthologs. To the extent that paralogsand/or nonorthologous gene displacements are present in the referencedmicroorganism that encode an enzyme catalyzing a similar orsubstantially similar metabolic reaction, those skilled in the art alsocan utilize these evolutionary related genes.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50;expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

In some embodiments, the present invention provides a non-naturallyoccurring microbial organism having a 1,3-butanediol (1,3-BDO) pathwaythat includes at least one exogenous nucleic acid encoding a 1,3-BDOpathway enzyme or protein expressed in a sufficient amount to produce1,3-BDO. The 1,3-BDO pathway includes Methanol methyltransferase (MtaB),Corrinoid protein (MtaC), Methyltetrahydrofolate:corrinoid proteinmethyltransferase (MtaA), Methyltetrahydrofolate:corrinoid proteinmethyltransferase (AcsE), Corrinoid iron-sulfur protein (AcsD),Nickel-protein assembly protein (AcsF & CooC), Ferredoxin (Orf7),Acetyl-CoA synthase (AcsB & AcsC), Carbon monoxide dehydrogenase (AcsA),Hydrogenase (Hyd), Acetoacetyl-CoA thiolase (AtoB), Acetoacetyl-CoAreductase (CoA-dependent, aldehyde forming), 3-oxobutyraldehydereductase (ketone reducing), 3-hydroxybutyraldehyde reductase,Acetoacetyl-CoA reductase (CoA-dependent, alcohol forming),3-oxobutyraldehyde reductase (aldehyde reducing), 4-hydroxy,2-butanonereductase, Acetoacetyl-CoA reductase (ketone reducing),3-hydroxybutyryl-CoA reductase (aldehyde forming), 3-hydroxybutyryl-CoAreductase (alcohol forming), 3-hydroxybutyryl-CoA transferase,3-hydroxybutyryl-CoA hydrolase, 3-hydroxybutyryl-CoA synthetase,3-hydroxybutyrate dehydrogenase, 3-hydroxybutyrate reductase,acetoacetyl-CoA transferase, acetoacetyl-CoA hydrolase, acetoacetyl-CoAsynthetase, or acetoacetate reductase.

In some embodiments, the 1,3-BDO pathway enzymes are a set of enzymesselected from: A: 1) Methanol methyltransferase (MtaB), 2) Corrinoidprotein (MtaC), 3) Methyltetrahydrofolate:corrinoid proteinmethyltransferase (MtaA), 4) Methyltetrahydrofolate:corrinoid proteinmethyltransferase (AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6)Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin (Orf7), 8)Acetyl-CoA synthase (AcsB & AcsC), 9) Carbon monoxide dehydrogenase(AcsA), 10) Hydrogenase, 11) Acetoacetyl-CoA thiolase (AtoB), 12)Acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming), 13)3-oxobutyraldehyde reductase (aldehyde reducing), and 14)4-hydroxy,2-butanone reductase; B: 1) Methanol methyltransferase (MtaB),2) Corrinoid protein (MtaC), 3) Methyltetrahydrofolate:corrinoid proteinmethyltransferase (MtaA), 4) Methyltetrahydrofolate:corrinoid proteinmethyltransferase (AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6)Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin (Orf7), 8)Acetyl-CoA synthase (AcsB & AcsC), 9) Carbon monoxide dehydrogenase(AcsA), 10) Hydrogenase, 11) Acetoacetyl-CoA thiolase (AtoB), 12)Acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming), 13)3-oxobutyraldehyde reductase (ketone reducing), and 14)3-hydroxybutyraldehyde reductase; C: 1) Methanol methyltransferase(MtaB), 2) Corrinoid protein (MtaC), 3) Methyltetrahydrofolate:corrinoidprotein methyltransferase (MtaA), 4) Methyltetrahydrofolate:corrinoidprotein methyltransferase (AcsE), 5) Corrinoid iron-sulfur protein(AcsD), 6) Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin(Orf7), 8) Acetyl-CoA synthase (AcsB & AcsC), 9) Carbon monoxidedehydrogenase (AcsA), 10) Hydrogenase, 11) Acetoacetyl-CoA thiolase(AtoB), 12) Acetoacetyl-CoA reductase (ketone reducing), 13)3-hydroxybutyryl-CoA reductase (aldehyde forming), and 14)3-hydroxybutyraldehyde reductase; D: 1) Methanol methyltransferase(MtaB), 2) Corrinoid protein (MtaC), 3) Methyltetrahydrofolate:corrinoidprotein methyltransferase (MtaA), 4) Methyltetrahydrofolate:corrinoidprotein methyltransferase (AcsE), 5) Corrinoid iron-sulfur protein(AcsD), 6) Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin(Orf7), 8) Acetyl-CoA synthase (AcsB & AcsC), 9) Carbon monoxidedehydrogenase (AcsA), 10) Hydrogenase, 11) Acetoacetyl-CoA thiolase(AtoB), 12) Acetoacetyl-CoA reductase (ketone reducing), and 13)3-hydroxybutyryl-CoA reductase (alcohol forming); E: 1) Methanolmethyltransferase (MtaB), 2) Corrinoid protein (MtaC), 3)Methyltetrahydrofolate:corrinoid protein methyltransferase (MtaA), 4)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 5)Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein(AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB &AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11)Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA reductase(CoA-dependent, alcohol forming), and 14) 4-hydroxy,2-butanonereductase; F: 1) Methanol methyltransferase (MtaB), 2) Corrinoid protein(MtaC), 3) Methyltetrahydrofolate:corrinoid protein methyltransferase(MtaA), 4) Methyltetrahydrofolate:corrinoid protein methyltransferase(AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6) Nickel-proteinassembly protein (AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoAsynthase (AcsB & AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10)Hydrogenase, 11) Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoAtransferase, hydrolase, or synthetase, 13) Acetoacetate reductase, 14)3-oxobutyraldehyde reductase (ketone reducing), and 15)3-hydroxybutyraldehyde reductase; G: 1) Methanol methyltransferase(MtaB), 2) Corrinoid protein (MtaC), 3) Methyltetrahydrofolate:corrinoidprotein methyltransferase (MtaA), 4) Methyltetrahydrofolate:corrinoidprotein methyltransferase (AcsE), 5) Corrinoid iron-sulfur protein(AcsD), 6) Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin(Orf7), 8) Acetyl-CoA synthase (AcsB & AcsC), 9) Carbon monoxidedehydrogenase (AcsA), 10) Hydrogenase, 11) Acetoacetyl-CoA thiolase(AtoB), 12) Acetoacetyl-CoA transferase, hydrolase, or synthetase, 13)Acetoacetate reductase, 14) 3-oxobutyraldehyde reductase (aldehydereducing), and 15) 4-hydroxy,2-butanone reductase; H: 1) Methanolmethyltransferase (MtaB), 2) Corrinoid protein (MtaC), 3)Methyltetrahydrofolate:corrinoid protein methyltransferase (MtaA), 4)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 5)Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein(AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB &AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11)Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA reductase (ketonereducing), 13) 3-hydroxybutyryl-CoA transferase, hydrolase, orsynthetase, 14) 3-hydroxybutyrate reductase, and 15)3-hydroxybutyraldehyde reductase; I: 1) Methanol methyltransferase(MtaB), 2) Corrinoid protein (MtaC), 3) Methyltetrahydrofolate:corrinoidprotein methyltransferase (MtaA), 4) Methyltetrahydrofolate:corrinoidprotein methyltransferase (AcsE), 5) Corrinoid iron-sulfur protein(AcsD), 6) Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin(Orf7), 8) Acetyl-CoA synthase (AcsB & AcsC), 9) Carbon monoxidedehydrogenase (AcsA), 10) Hydrogenase, 11) Acetoacetyl-CoA thiolase(AtoB), 12) Acetoacetyl-CoA transferase, hydrolase, or synthetase, 13)3-hydroxybutyrate dehydrogenase, 14) 3-hydroxybutyrate reductase, and15) 3-hydroxybutyraldehyde reductase.

The non-naturally occurring microbial organism can include two exogenousnucleic acids each encoding a 1,3-BDO pathway enzyme, in someembodiments, three exogenous nucleic acids each encoding a 1,3-BDOpathway enzyme, in other embodiments, four exogenous nucleic acids eachencoding a 1,3-BDO pathway enzyme, in other embodiments, five exogenousnucleic acids each encoding a 1,3-BDO pathway enzyme, in otherembodiments, six exogenous nucleic acids each encoding a 1,3-BDO pathwayenzyme in other embodiments, seven exogenous nucleic acids each encodinga 1,3-BDO pathway enzyme, in other embodiments, eight exogenous nucleicacids each encoding a 1,3-BDO pathway enzyme, in other embodiments, nineexogenous nucleic acids each encoding a 1,3-BDO pathway enzyme, in otherembodiments, 10 exogenous nucleic acids each encoding a 1,3-BDO pathwayenzyme, in other embodiments, 11 exogenous nucleic acids each encoding a1,3-BDO pathway enzyme, in other embodiments, 12 exogenous nucleic acidseach encoding a 1,3-BDO pathway enzyme, in other embodiments, 13exogenous nucleic acids each encoding a 1,3-BDO pathway enzyme, in otherembodiments, 14 exogenous nucleic acids each encoding a 1,3-BDO pathwayenzyme, in other embodiments, 15 exogenous nucleic acids each encoding a1,3-BDO pathway enzyme, in still other embodiments. Any of the at leastone exogenous nucleic acid can be a heterologous nucleic acid and thenon-naturally occurring microbial organism can be constructed forculturing in a substantially anaerobic culture medium. Such microbialorganisms can use a carbon feedstock selected from 1) methanol and CO,2) methanol, CO₂, and H₂, 3) methanol, CO, CO₂, and H₂, 4) methanol andsynthesis gas comprising CO and H₂, 5) methanol and synthesis gascomprising CO, CO₂, and H₂, 6) one or more carbohydrates, 7) methanoland one or more carbohydrates, and 8) methanol. Exemplary carbohydratesinclude, but are not limited to, glucose, sucrose, xylose, arabinose,and glycerol.

In some embodiments, the present invention provides a non-naturallyoccurring microbial organism, comprising a microbial organism having a1,3-butanediol (1,3-BDO) pathway comprising at least one exogenousnucleic acid encoding a 1,3-BDO pathway enzyme or protein expressed in asufficient amount to produce 1,3-BDO said 1,3-BDO pathway comprisingFormate dehydrogenase, Formyltetrahydrofolate synthetase,Methenyltetrahydrofolate cyclohydrolase, Methylenetetrahydrofolatedehydrogenase, Methylenetetrahydrofolate reductase,Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE),Corrinoid iron-sulfur protein (AcsD), Nickel-protein assembly protein(AcsF & CooC), Ferredoxin (Orf7), Acetyl-CoA synthase (AcsB & AcsC),Carbon monoxide dehydrogenase (AcsA), Hydrogenase (Hyd), Acetoacetyl-CoAthiolase (AtoB), Acetoacetyl-CoA reductase (CoA-dependent, aldehydeforming), 3-oxobutyraldehyde reductase (ketone reducing),3-hydroxybutyraldehyde reductase, Acetoacetyl-CoA reductase(CoA-dependent, alcohol forming), 3-oxobutyraldehyde reductase (aldehydereducing), 4-hydroxy,2-butanone reductase, Acetoacetyl-CoA reductase(ketone reducing), 3-hydroxybutyryl-CoA reductase (aldehyde forming),3-hydroxybutyryl-CoA reductase (alcohol forming), 3-hydroxybutyryl-CoAtransferase, 3-hydroxybutyryl-CoA hydrolase, 3-hydroxybutyryl-CoAsynthetase, 3-hydroxybutyrate dehydrogenase, 3-hydroxybutyratereductase, acetoacetyl-CoA transferase, acetoacetyl-CoA hydrolase,acetoacetyl-CoA synthetase, or acetoacetate reductase.

In some embodiments, the 1,3-BDO pathway enzymes are a set of enzymesselected from: A: 1) Formate dehydrogenase, 2) Formyltetrahydrofolatesynthetase, 3) Methenyltetrahydrofolate cyclohydrolase, 4)Methylenetetrahydrofolate dehydrogenase, 5) Methylenetetrahydrofolatereductase, 6) Methyltetrahydrofolate:corrinoid protein methyltransferase(AcsE), 7) Corrinoid iron-sulfur protein (AcsD), 8) Nickel-proteinassembly protein (AcsF & CooC), 9) Ferredoxin (Orf7), 10) Acetyl-CoAsynthase (AcsB & AcsC), 11) Carbon monoxide dehydrogenase (AcsA), 12)Hydrogenase (Hyd), 13) Acetoacetyl-CoA thiolase (AtoB), 14)Acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming), 15)3-oxobutyraldehyde reductase (aldehyde reducing), and 16)4-hydroxy,2-butanone reductase; B: 1) Formate dehydrogenase, 2)Formyltetrahydrofolate synthetase, 3) Methenyltetrahydrofolatecyclohydrolase, 4) Methylenetetrahydrofolate dehydrogenase, 5)Methylenetetrahydrofolate reductase, 6) Methyltetrahydrofolate:corrinoidprotein methyltransferase (AcsE), 7) Corrinoid iron-sulfur protein(AcsD), 8) Nickel-protein assembly protein (AcsF & CooC), 9) Ferredoxin(Orf7), 10) Acetyl-CoA synthase (AcsB & AcsC), 11) Carbon monoxidedehydrogenase (AcsA), 12) Hydrogenase (Hyd), 13) Acetoacetyl-CoAthiolase (AtoB), 14) Acetoacetyl-CoA reductase (CoA-dependent, aldehydeforming), 15) 3-oxobutyraldehyde reductase (ketone reducing), and 16)3-hydroxybutyraldehyde reductase; C: 1) Formate dehydrogenase, 2)Formyltetrahydrofolate synthetase, 3) Methenyltetrahydrofolatecyclohydrolase, 4) Methylenetetrahydrofolate dehydrogenase, 5)Methylenetetrahydrofolate reductase, 6) Methyltetrahydrofolate:corrinoidprotein methyltransferase (AcsE), 7) Corrinoid iron-sulfur protein(AcsD), 8) Nickel-protein assembly protein (AcsF & CooC), 9) Ferredoxin(Orf7), 10) Acetyl-CoA synthase (AcsB & AcsC), 11) Carbon monoxidedehydrogenase (AcsA), 12) Hydrogenase (Hyd), 13) Acetoacetyl-CoAthiolase (AtoB), 14) Acetoacetyl-CoA reductase (ketone reducing), 15)3-hydroxybutyryl-CoA reductase (aldehyde forming), and 16)3-hydroxybutyraldehyde reductase; D: 1) Formate dehydrogenase, 2)Formyltetrahydrofolate synthetase, 3) Methenyltetrahydrofolatecyclohydrolase, 4) Methylenetetrahydrofolate dehydrogenase, 5)Methylenetetrahydrofolate reductase, 6) Methyltetrahydrofolate:corrinoidprotein methyltransferase (AcsE), 7) Corrinoid iron-sulfur protein(AcsD), 8) Nickel-protein assembly protein (AcsF & CooC), 9) Ferredoxin(Orf7), 10) Acetyl-CoA synthase (AcsB & AcsC), 11) Carbon monoxidedehydrogenase (AcsA), 12) Hydrogenase (Hyd), 13) Acetoacetyl-CoAthiolase (AtoB), 14) Acetoacetyl-CoA reductase (CoA-dependent, alcoholforming), and 15) 4-hydroxy,2-butanone reductase; E: 1) Formatedehydrogenase, 2) Formyltetrahydrofolate synthetase, 3)Methenyltetrahydrofolate cyclohydrolase, 4) Methylenetetrahydrofolatedehydrogenase, 5) Methylenetetrahydrofolate reductase, 6)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 7)Corrinoid iron-sulfur protein (AcsD), 8) Nickel-protein assembly protein(AcsF & CooC), 9) Ferredoxin (Orf7), 10) Acetyl-CoA synthase (AcsB &AcsC), 11) Carbon monoxide dehydrogenase (AcsA), 12) Hydrogenase (Hyd),13) Acetoacetyl-CoA thiolase (AtoB), 14) Acetoacetyl-CoA reductase(ketone reducing), and 15) 3-hydroxybutyryl-CoA reductase (alcoholforming); F: 1) Formate dehydrogenase, 2) Formyltetrahydrofolatesynthetase, 3) Methenyltetrahydrofolate cyclohydrolase, 4)Methylenetetrahydrofolate dehydrogenase, 5) Methylenetetrahydrofolatereductase, 6) Methyltetrahydrofolate:corrinoid protein methyltransferase(AcsE), 7) Corrinoid iron-sulfur protein (AcsD), 8) Nickel-proteinassembly protein (AcsF & CooC), 9) Ferredoxin (Orf7), 10) Acetyl-CoAsynthase (AcsB & AcsC), 11) Carbon monoxide dehydrogenase (AcsA), 12)Hydrogenase (Hyd), 13) Acetoacetyl-CoA thiolase (AtoB), 14)Acetoacetyl-CoA transferase, hydrolase, or synthetase, 15) Acetoacetatereductase, 16) 3-oxobutyraldehyde reductase (ketone reducing), and 17)3-hydroxybutyraldehyde reductase; G: 1) Formate dehydrogenase, 2)Formyltetrahydrofolate synthetase, 3) Methenyltetrahydrofolatecyclohydrolase, 4) Methylenetetrahydrofolate dehydrogenase, 5)Methylenetetrahydrofolate reductase, 6) Methyltetrahydrofolate:corrinoidprotein methyltransferase (AcsE), 7) Corrinoid iron-sulfur protein(AcsD), 8) Nickel-protein assembly protein (AcsF & CooC), 9) Ferredoxin(Orf7), 10) Acetyl-CoA synthase (AcsB & AcsC), 11) Carbon monoxidedehydrogenase (AcsA), 12) Hydrogenase (Hyd), 13) Acetoacetyl-CoAthiolase (AtoB), 14) Acetoacetyl-CoA transferase, hydrolase, orsynthetase, 15) Acetoacetate reductase, 16) 3-oxobutyraldehyde reductase(aldehyde reducing), and 17) 4-hydroxy,2-butanone reductase; H: 1)Formate dehydrogenase, 2) Formyltetrahydrofolate synthetase, 3)Methenyltetrahydrofolate cyclohydrolase, 4) Methylenetetrahydrofolatedehydrogenase, 5) Methylenetetrahydrofolate reductase, 6)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 7)Corrinoid iron-sulfur protein (AcsD), 8) Nickel-protein assembly protein(AcsF & CooC), 9) Ferredoxin (Orf7), 10) Acetyl-CoA synthase (AcsB &AcsC), 11) Carbon monoxide dehydrogenase (AcsA), 12) Hydrogenase (Hyd),13) Acetoacetyl-CoA thiolase (AtoB), 14) Acetoacetyl-CoA reductase(ketone reducing), 15) 3-hydroxybutyryl-CoA transferase, hydrolase, orsynthetase, 16) 3-hydroxybutyrate reductase, and 17)3-hydroxybutyraldehyde reductase; I: 1) Formate dehydrogenase, 2)Formyltetrahydrofolate synthetase, 3) Methenyltetrahydrofolatecyclohydrolase, 4) Methylenetetrahydrofolate dehydrogenase, 5)Methylenetetrahydrofolate reductase, 6) Methyltetrahydrofolate:corrinoidprotein methyltransferase (AcsE), 7) Corrinoid iron-sulfur protein(AcsD), 8) Nickel-protein assembly protein (AcsF & CooC), 9) Ferredoxin(Orf7), 10) Acetyl-CoA synthase (AcsB & AcsC), 11) Carbon monoxidedehydrogenase (AcsA), 12) Hydrogenase (Hyd), 13) Acetoacetyl-CoAthiolase (AtoB), 14) Acetoacetyl-CoA transferase, hydrolase, orsynthetase, 15) 3-hydroxybutyrate dehydrogenase, 16) 3-hydroxybutyratereductase, and 17) 3-hydroxybutyraldehyde reductase.

The non-naturally occurring microbial organism can include two exogenousnucleic acids each encoding a 1,3-BDO pathway enzyme, in someembodiments, three exogenous nucleic acids each encoding a 1,3-BDOpathway enzyme, in other embodiments, four exogenous nucleic acids eachencoding a 1,3-BDO pathway enzyme, in other embodiments, five exogenousnucleic acids each encoding a 1,3-BDO pathway enzyme, in otherembodiments, six exogenous nucleic acids each encoding a 1,3-BDO pathwayenzyme in other embodiments, seven exogenous nucleic acids each encodinga 1,3-BDO pathway enzyme, in other embodiments, eight exogenous nucleicacids each encoding a 1,3-BDO pathway enzyme, in other embodiments, nineexogenous nucleic acids each encoding a 1,3-BDO pathway enzyme, in otherembodiments, 10 exogenous nucleic acids each encoding a 1,3-BDO pathwayenzyme, in other embodiments, 11 exogenous nucleic acids each encoding a1,3-BDO pathway enzyme, in other embodiments, 12 exogenous nucleic acidseach encoding a 1,3-BDO pathway enzyme, in other embodiments, 13exogenous nucleic acids each encoding a 1,3-BDO pathway enzyme, in otherembodiments, 14 exogenous nucleic acids each encoding a 1,3-BDO pathwayenzyme, in other embodiments, 15 exogenous nucleic acids each encoding a1,3-BDO pathway enzyme, 16 exogenous nucleic acids each encoding a1,3-BDO pathway enzyme, in other embodiments, and 17 exogenous nucleicacids, in still other embodiments. Any of the at least one exogenousnucleic acid can be a heterologous nucleic acid and the non-naturallyoccurring microbial organism can be constructed for culturing in asubstantially anaerobic culture medium. Such microbial organisms can usea carbon feedstock selected from 1) CO, 2) CO₂ and H₂, 3) CO, CO₂, andH₂, 4) synthesis gas comprising CO and H₂, 5) synthesis gas comprisingCO, CO₂, and H₂, and 6) one or more carbohydrates. Exemplarycarbohydrates include, but are not limited to, glucose, sucrose, xylose,arabinose, and glycerol.

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organisms having a 1,3-butanediol pathways, whereinthe non-naturally occurring microbial organism comprises at least oneexogenous nucleic acid encoding an enzyme or protein that converts asubstrate to a product selected from the group consisting of carbondioxide to FOR, FOR to 10FTHF, 10FTHF to METHF, METHF to MLTHF, MLTHF to5MTHF, methanol to CH₃-MtaC, CH₃-MtaC to 5MTHF, 5MTHF to CFeSp-CH₃,CFeSp-CH₃ to acetyl-CoA, acetyl-CoA to acetoacetyl-CoA, acetoacetyl-CoAto 3-hydroxybutryl-CoA, 3-hydroxybutryl-CoA to 3-hydroxybutyraldehyde,3-hydroxybutryaldehyde to 1,3-BDO, 3-hydroxybutryl-CoA to 1,3-BDO,acetoacetyl-CoA to 4-hydroxy-2 butanone, acetoacetyl-CoA to3-oxobutryaldehyde, 3-oxobutryaldehyde to 4-hydroxy-2-butanone,4-hydroxy-2-butanone to 1,3-BDO, 3-oxobutyraldehdye to3-hydroxybutyraldehyde, acetoacetyl-CoA to acetoacetate, acetoacetate to3-oxobutyraldehyde, acetoacetate to 3-hydroxybutyrate,3-hydroxybutyryl-CoA to 3-hydroxybutyrate, and 3-hydroxybutyrate to3-hydroxybutyraldehyde. One skilled in the art will understand thatthese are merely exemplary and that any of the substrate-product pairsdisclosed herein suitable to produce a desired product and for which anappropriate activity is available for the conversion of the substrate tothe product can be readily determined by one skilled in the art based onthe teachings herein. Thus, the invention provides a non-naturallyoccurring microbial organism containing at least one exogenous nucleicacid encoding an enzyme or protein, where the enzyme or protein convertsthe substrates and products of a 1,3-butanediol pathway, such as thoseshown in FIGS. 4 and 5.

While generally described herein as a microbial organism that contains a1,3-butanediol pathway, it is understood that the invention additionallyprovides a non-naturally occurring microbial organism comprising atleast one exogenous nucleic acid encoding a 1,3-butanediol pathwayenzyme or protein expressed in a sufficient amount to produce anintermediate of a 1,3-butanediol pathway. For example, as disclosedherein, 1,3-butanediol pathway are exemplified in FIGS. 4 and 5.Therefore, in addition to a microbial organism containing a1,3-butanediol pathway that produces 1,3-butanediol, the inventionadditionally provides a non-naturally occurring microbial organismcomprising at least one exogenous nucleic acid encoding a 1,3-butanediolpathway enzyme, where the microbial organism produces a 1,3-butanediolpathway intermediate, for example, acetyl-CoA, acetoacetyl-CoA,acetoacetate, 3-hydroxybutryl-CoA, 3-hydroxybutyrate,3-oxobutyraldehyde, 4-hydroxy-2-butanone, and 3-hydroxybutryaldehyde.

It is understood that any of the pathways disclosed herein, as describedin the Examples and exemplified in the Figures, including the pathwaysof FIGS. 4 and 5, can be utilized to generate a non-naturally occurringmicrobial organism that produces any pathway intermediate or product, asdesired. As disclosed herein, such a microbial organism that produces anintermediate can be used in combination with another microbial organismexpressing downstream pathway enzymes to produce a desired product.However, it is understood that a non-naturally occurring microbialorganism that produces a 1,3-butanediol pathway intermediate can beutilized to produce the intermediate as a desired product. Thus, thenon-naturally occurring organisms of the invention can be used toproduce, for example, acetoacetate, 3-hydroxybutyrate,3-oxobutyraldehyde, 3-hydroxybutyraldehyde, or 4-hydroxy-2-butanone.Thus, any of these intermediate products can be produced in a separateorganism utilizing the pathways shown in FIGS. 4 and 5.

In some embodiments, non-naturally occurring organisms described hereinhave three capabilities which are depicted in FIG. 4: 1) a functionalmethyltransferase system allowing the production of5-methyl-tetrahydrofolate (Me-THF) from methanol and THF, 2) the abilityto combine CO, Coenzyme A, and the methyl group of Me-THF to formacetyl-CoA, and 3) the ability to synthesize 1,3-butanediol fromacetyl-CoA. The latter can be facilitated by the generation of pyruvatefrom a carbohydrate via glycolysis. Glycolysis is an anaerobic metabolicpathway that is found in the cytoplasm of cells in all living organisms.The process converts one molecule of glucose into two molecules ofpyruvate, while providing two net molecules of ATP:Glucose+2NAD⁺+2P_(i)+2ADP→2pyruvate+2NADH+2ATP+2H⁺+2H₂O

Such non-naturally occurring organisms can ‘fix’ carbon from exogenousCO and/or CO₂ and methanol to synthesize acetyl-CoA, cell mass, andproducts. Note that implementing the pathway to form acetyl-CoA frommethanol and syngas is energetically favorable compared to utilizing thefull Wood-Ljungdahl pathway. For example, the direct conversion ofsynthesis gas to acetate is an energetically neutral process (see FIGS.1 and 2). Specifically, one ATP molecule is consumed during theformation of formyl-THF by formyl-THF synthase and one ATP molecule isproduced during the production of acetate via acetate kinase. Thepathways disclosed herein circumvent the ATP consumption by ensuringthat the methyl group on the methyl branch product, methyl-THF, isobtained from methanol rather than CO₂. This ensures that acetateformation has a positive ATP yield that can help support cell growth andmaintenance. A host organism engineered with these capabilities thatalso possesses the capability for anapleurosis (e.g., E. coli) can growon the methanol and syngas-generated acetyl-CoA in the presence of asuitable external electron acceptor such as nitrate. This electronacceptor is used to accept electrons from the reduced quinone formed viasuccinate dehydrogenase. A further benefit of adding an externalelectron acceptor is that additional energy for cell growth,maintenance, and product formation can be generated from respiration ofacetyl-CoA. In some embodiments, another non-naturally occurringmicrobial organism has a pyruvate ferredoxin oxidoreductase (PFOR)enzyme or other enzymes that facilitate the conversion of pyruvate intoacetyl-CoA or vice versa into the strain to facilitate the synthesis ofbiomass precursors in the absence of an external electron acceptor. Afurther characteristic of such non-naturally occurring organisms is thecapability for extracting reducing equivalents from molecular hydrogen.This enables a high yield of reduced products such as ethanol, butanol,isobutanol, isopropanol, 1,4-butanediol, 1,3-butanediol, succinic acid,fumaric acid, malic acid, 4-hydroxybutyric acid, 3-hydroxypropionicacid, lactic acid, adipic acid, methacrylic acid, and acrylic acid.

The organisms described in this invention can produce acetyl-CoA, cellmass, and targeted chemicals, more specifically 1,3-BDO, from: 1)methanol and CO, 2) methanol, CO₂, and H₂, 3) methanol, CO, CO₂, and H₂,4) methanol and synthesis gas comprising CO and H₂, 5) methanol andsynthesis gas comprising CO, CO₂, and H₂, 6) one or more carbohydrates,7) methanol and one or more carbohydrates, and 8) methanol. Exemplarycarbohydrates include, but are not limited to, glucose, sucrose, xylose,arabinose, and glycerol.

Successfully engineering these pathways into an organism involvesidentifying an appropriate set of enzymes, cloning their correspondinggenes into a production host, optimizing the stability and expression ofthese genes, optimizing fermentation conditions, and assaying forproduct formation following fermentation. A number of enzymes thatcatalyze each step of the pathways used the conversion of synthesis gasand methanol to acetyl-CoA, and further to 1,3-butanediol are describedbelow. To engineer a production host for the utilization of syngas andmethanol, one or more exogenous DNA sequence(s) encoding the enzymes ofthese pathways can be expressed in the microorganism.

Expression of the modified Wood-Ljungdahl pathway in a foreign host, asshown in FIG. 4, involves introducing a set of methyltransferases toutilize the carbon and hydrogen provided by methanol and the carbonprovided by CO and/or CO₂. A complex of 3 methyltransferase proteins,denoted MtaA, MtaB, and MtaC, perform the desired methanolmethyltransferase activity (Sauer et al., Eur. J. Biochem. 243.3:670-677(1997); Naidu and Ragsdale, J. Bacteriol. 183.11:3276-3281 (2001);Tallant and Krzycki, J. Biol. Chem. 276.6:4485-4493 (2001); Tallant andKrzycki, J. Bacteriol. 179.22:6902-6911 (1997); Tallant and Krzycki, J.Bacteriol. 178.5:1295-1301 (1996); Ragsdale, S. W., Crit. Rev. Biochem.Mol. Biol. 39.3:165-195 (2004).

MtaB is a zinc protein that catalyzes the transfer of a methyl groupfrom methanol to MtaC, a corrinoid protein. Exemplary genes encodingMtaB and MtaC can be found in methanogenic archaea such asMethanosarcina barkeri (Maeder et al., J. Bacteriol. 188.22:7922-7931(2006)) and Methanosarcina acetivorans (Galagan et al., Genome Res.12/4:532-542 (2002)), as well as the acetogen, Moorella thermoacetica(Das et al., Proteins 67.1:167-176 (2007)). In general, the MtaB andMtaC genes are adjacent to one another on the chromosome as theiractivities are tightly interdependent. The protein sequences of variousMtaB and MtaC encoding genes in M. barkeri, M. acetivorans, and M.thermoaceticum can be identified by their following GenBank accessionnumbers:

Protein GenBank ID GI number Organism MtaB1 YP_304299 73668284Methanosarcina barkeri MtaC1 YP_304298 73668283 Methanosarcina barkeriMtaB2 YP_307082 73671067 Methanosarcina barkeri MtaC2 YP_307081 73671066Methanosarcina barkeri MtaB3 YP_304612 73668597 Methanosarcina barkeriMtaC3 YP_304611 73668596 Methanosarcina barkeri MtaB1 NP_615421 20089346Methanosarcina acetivorans MtaB1 NP_615422 20089347 Methanosarcinaacetivorans MtaB2 NP_619254 20093179 Methanosarcina acetivorans MtaC2NP_619253 20093178 Methanosarcina acetivorans MtaB3 NP_616549 20090474Methanosarcina acetivorans MtaC3 NP_616550 20090475 Methanosarcinaacetivorans MtaB YP_430066 83590057 Moorella thermoacetica MtaCYP_430065 83590056 Moorella thermoacetica

The MtaB1 and MtaC1 genes, YP_(—)304299 and YP_(—)304298, from M.barkeri were cloned into E. coli and sequenced (Sauer et al., Eur. J.Biochem. 243.3:670-677 (1997)). The crystal structure of thismethanol-cobalamin methyltransferase complex is also available Hagemeieret al., Proc. Natl. Acad. Sci. U.S.A. 103.50:18917-18922 (2006)). TheMtaB genes, YP_(—)307082 and YP_(—)304612, in M. barkeri were identifiedby sequence homology to YP_(—)304299. In general, homology searches arean effective means of identifying methanol methyltransferases becauseMtaB encoding genes show little or no similarity to methyltransferasesthat act on alternative substrates such as trimethylamine,dimethylamine, monomethylamine, or dimethylsulfide. The MtaC genes,YP_(—)307081 and YP_(—)304611, were identified based on their proximityto the MtaB genes and also their homology to YP_(—)304298. The threesets of MtaB and MtaC genes from M. acetivorans have been genetically,physiologically, and biochemically characterized Pritchett and Metcalf,Mol. Microbiol. 56.5:1183-1194 (2005)). Mutant strains lacking two ofthe sets were able to grow on methanol, whereas a strain lacking allthree sets of MtaB and MtaC genes sets could not grow on methanol. Thisindicates that each set of genes plays a role in methanol utilization.The M. thermoacetica MtaB gene was identified based on homology to themethanogenic MtaB genes and also by its adjacent chromosomal proximityto the methanol-induced corrinoid protein, MtaC, which has beencrystallized (Zhou et al., Acta Crystallogr. Sect. F Struct. Biol Cryst.Commun. 61 Pt. 5:537-540 (2005)) and further characterized by Northernhybridization and Western Blotting (Das et al. Proteins 67.1:167-76(2007)).

MtaA is zinc protein that catalyzes the transfer of the methyl groupfrom MtaC to either Coenzyme M in methanogens or methyltetrahydrofolatein acetogens. MtaA can also utilize methylcobalamin as the methyl donor.Exemplary genes encoding MtaA can be found in methanogenic archaea suchas Methanosarcina barkeri (Maeder et al., J. Bacteriol. 188.22 7922-7931(2006)) and Methanosarcina acetivorans (Galagan et al., Genome Res.12.4:532-542 (2002)), as well as the acetogen, Moorella thermoacetica(Das et al., Proteins 67.1:167-176 (2007)). In general, MtaA proteinsthat catalyze the transfer of the methyl group from CH₃-MtaC aredifficult to identify bioinformatically as they share similarity toother corrinoid protein methyltransferases and are not oriented adjacentto the MtaB and MtaC genes on the chromosomes. Nevertheless, a number ofMtaA encoding genes have been characterized. The protein sequences ofthese genes in M. barkeri and M. acetivorans can be identified by thefollowing GenBank accession numbers:

Protein GenBank ID GI number Organism MtaA YP_304602 73668587Methanosarcina barkeri MtaA1 NP_619241 20093166 Methanosarcinaacetivorans MtaA2 NP_616548 20090473 Methanosarcina acetivorans

The MtaA gene, YP_(—)304602, from M. barkeri was cloned, sequenced, andfunctionally overexpressed in E. coli (Harms and Thauer, Eur. J.Biochem. 135.3:653-659 (1996). In M. acetivorans, MtaA1 is used forgrowth on methanol, whereas MtaA2 is dispensable even though methaneproduction from methanol is reduced in MtaA2 mutants (Bose et al. J.Bacteriol. 190.11:4017-4026 (2008). Moreover, there are multipleadditional MtaA homologs in M. barkeri and M. acetivorans that are asyet uncharacterized, but can also catalyze corrinoid proteinmethyltransferase activity.

Putative MtaA encoding genes in M. thermoacetica were identified bytheir sequence similarity to the characterized methanogenic MtaA genes.Specifically, three M. thermoacetica genes show high homology (>30%sequence identity) to YP_(—)304602 from M. barkeri. Unlike methanogenicMtaA proteins that naturally catalyze the transfer of the methyl groupfrom CH₃-MtaC to Coenzyme M, an M. thermoacetica MtaA can transfer themethyl group to methyltetrahydrofolate given the similar roles ofmethyltetrahydrofolate and Coenzyme M in methanogens and acetogens,respectively. The protein sequences of putative MtaA encoding genes fromM. thermoacetica can be identified by the following GenBank accessionnumbers:

GenBank Protein ID GI number Organism MtaA YP_430937 83590928 Moorellathermoacetica MtaA YP_431175 83591166 Moorella thermoacetica MtaAYP_430935 83590926 Moorella thermoacetica

ACS/CODH is the central enzyme of the carbonyl branch of theWood-Ljungdahl pathway. It catalyzes the reversible reduction of carbondioxide to carbon monoxide and also the synthesis of acetyl-CoA fromcarbon monoxide, Coenzyme A, and the methyl group from a methylatedcorrinoid-iron-sulfur protein. The corrinoid-iron-sulfur-protein ismethylated by methyltetrahydrofolate via a methyltransferase. Expressionof ACS/CODH in a foreign host involves introducing one or more of thefollowing proteins: Methyltetrahydrofolate:corrinoid, proteinmethyltransferase (AcsE), Corrinoid iron-sulfur protein (AcsD),Nickel-protein assembly protein (AcsF), Ferredoxin (Orf7), Acetyl-CoAsynthase (AcsB and AcsC), Carbon monoxide dehydrogenase (AcsA),Nickel-protein assembly protein (CooC).

The genes used for carbon-monoxide dehydrogenase/acetyl-CoA synthaseactivity typically reside in a limited region of the native genome thatmay be an extended operon (Ragsdale, S. W., Crit. Rev. Biochem. Mol.Biol. 39.3:165-195 (2004); Morton et al., J. Biol. Chem.266.35:23824-23828 (1991); Roberts et al., Proc. Natl. Acad. Sci. U.S.A.86.1:32-36 (1989)). Each of the genes in this operon from the acetogen,M. thermoacetica, has already been cloned and expressed actively in E.coli (Morton et al., supra (1991); Roberts et al., supra (1989); Lu etal., J. Biol. Chem. 268.8:5605-5614 91993)). The protein sequences ofthese genes can be identified by the following GenBank accessionnumbers:

Protein GenBank ID GI number Organism AcsE YP_430054 83590045 Moorellathermoacetica AcsD YP_430055 83590046 Moorella thermoacetica AcsFYP_430056 83590047 Moorella thermoacetica Orf7 YP_430057 83590048Moorella thermoacetica AcsC YP_430058 83590049 Moorella thermoaceticaAcsB YP_430059 83590050 Moorella thermoacetica AcsA YP_430060 83590051Moorella thermoacetica CooC YP_430061 83590052 Moorella thermoacetica

The hydrogenogenic bacterium, Carboxydothermus hydrogenoformans, canutilize carbon monoxide as a growth substrate by means of acetyl-CoAsynthase (Wu et al. PLos Genet. 1.5:e65 (2005)). In strain Z-2901, theacetyl-CoA synthase enzyme complex lacks carbon monoxide dehydrogenasedue to a frameshift mutation (Wu et al. PLos Genet. 1.5:e65 (2005)),whereas in strain DSM 6008, a functional unframeshifted full-lengthversion of this protein has been purified (Svetlitchnyi et al., Proc.Natl. Acad. Sci. U.S.A. 101.2:446-451 (2004)). The protein sequences ofthe C. hydrogenoformans genes from strain Z-2901 can be identified bythe following GenBank accession numbers:

Protein GenBank ID GI number Organism AcsE YP_360065 78044202Carboxydothermus hydrogenoformans AcsD YP_360064 78042962Carboxydothermus hydrogenoformans AcsF YP_360063 78044060Carboxydothermus hydrogenoformans Orf7 YP_360062 78044449Carboxydothermus hydrogenoformans AcsC YP_360061 78043584Carboxydothermus hydrogenoformans AcsB YP_360060 78042742Carboxydothermus hydrogenoformans CooC YP_360059 78044249Carboxydothermus hydrogenoformans

The methanogenic archaeon, Methanosarcina acetivorans, can also grow oncarbon monoxide, exhibits acetyl-CoA synthase/carbon monoxidedehydrogenase activity, and produces both acetate and formate (Lessneret al., Proc. Natl. Acad. Sci. U.S.A. 103.47:17921-17926 (2006)). Thisorganism contains two sets of genes that encode ACS/CODH activity(Rother and Metcalf, Proc. Natl. Acad. Sci. U.S.A. 101.48:16929-16934(2004)). The protein sequences of both sets of M. acetivorans genes canbe identified by the following GenBank accession numbers:

Protein GenBank ID GI number Organism AcsC NP_618736 20092661Methanosarcina acetivorans AcsD NP_618735 20092660 Methanosarcinaacetivorans AcsF, CooC NP_618734 20092659 Methanosarcina acetivoransAcsB NP_618733 20092658 Methanosarcina acetivorans AcsEps NP_61873220092657 Methanosarcina acetivorans AcsA NP_618731 20092656Methanosarcina acetivorans AcsC NP_615961 20089886 Methanosarcinaacetivorans AcsD NP_615962 20089887 Methanosarcina acetivorans AcsF,CooC NP_615963 20089888 Methanosarcina acetivorans AcsB NP_61596420089889 Methanosarcina acetivorans AcsEps NP_615965 20089890Methanosarcina acetivorans AcsA NP_615966 20089891 Methanosarcinaacetivorans

The AcsC, AcsD, AcsB, AcsE, and AcsA proteins are commonly referred toas the gamma, delta, beta, epsilon, and alpha subunits of themethanogenic CODH/ACS. Homologs to the epsilon encoding genes are notpresent in acetogens such as M. thermoacetica or hydrogenogenic bacteriasuch as C. hydrogenoformans. Hypotheses for the existence of two activeCODH/ACS operons in M. acetivorans include catalytic properties (i.e.,K_(m), V_(max), k_(at)) that favor carboxidotrophic or aceticlasticgrowth or differential gene regulation enabling various stimuli toinduce CODH/ACS expression (Rother et al., Arch. Microbiol.188.5:463-472 (2007)).

In both M. thermoacetica and C. hydrogenoformans, additional CODHencoding genes are located outside of the ACS/CODH operons. Theseenzymes provide a means for extracting electrons (or reducingequivalents) from the conversion of carbon monoxide to carbon dioxide.The reducing equivalents are then passed to acceptors such as oxidizedferredoxin, NADP+, water, or hydrogen peroxide to form reducedferredoxin, NADPH, H₂, or water, respectively. In some cases,hydrogenase encoding genes are located adjacent to a CODH. InRhodospirillum rubrum, the encoded CODH/hydrogenase proteins form amembrane-bound enzyme complex that has been indicated to be a site whereenergy, in the form of a proton gradient, is generated from theconversion of CO to CO₂ and H₂ (Fox et al., J. Bacteriol.178.21:6200-6208 (1996)). The CODH-I of C. hydrogenoformans and itsadjacent genes have been proposed to catalyze a similar functional rolebased on their similarity to the R. rubrum CODH/hydrogenase gene cluster(Wu et al., PLoS Genet. 1.5:e65 (2005)). The C. hydrogenoformans CODH-Iwas also shown to exhibit intense CO oxidation and CO₂ reductionactivities when linked to an electrode (Parkin et al., J. Am. Chem.Soc., 129.34:10328-10329 (2007)). The genes encoding the C.hydrogenoformans CODH-II and CooF, a neighboring protein, were clonedand sequenced (Gonzalez and Robb, FEMS Microb. Lett. 191.2:243-247(2000)). The resulting complex was membrane-bound, although cytoplasmicfractions of CODH-II were shown to catalyze the formation of NADPHsuggesting an anabolic role (Svetlitchnyi et al., J. Bacteriol.183.17:5134-5144 (2001)). The crystal structure of the CODH-II is alsoavailable (Dobbek et al., Science 293.5533:1281-1285 (2001)). Theprotein sequences of exemplary CODH and hydrogenase genes can beidentified by the following GenBank accession numbers:

Protein GenBank ID GI number Organism CODH (putative) YP_430813 83590804Moorella thermoacetica CODH-I (CooS-I) YP_360644 78043418Carboxydothermus hydrogenoformans CooF YP_360645 78044791Carboxydothermus hydrogenoformans HypA YP_360646 78044340Carboxydothermus hydrogenoformans CooH YP_360647 78043871Carboxydothermus hydrogenoformans CooU YP_360648 78044023Carboxydothermus hydrogenoformans CooX YP_360649 78043124Carboxydothermus hydrogenoformans CooL YP_360650 78043938Carboxydothermus hydrogenoformans CooK YP_360651 78044700Carboxydothermus hydrogenoformans CooM YP_360652 78043942Carboxydothermus hydrogenoformans CooM AAC45116 1515466 Rhodospirillumrubrum CooK AAC45117 1515467 Rhodospirillum rubrum CooL AAC45118 1515468Rhodospirillum rubrum CooX AAC45119 1515469 Rhodospirillum rubrum CooUAAC45120 1515470 Rhodospirillum rubrum CooH AAC45121 1498746Rhodospirillum rubrum CooF AAC45122 1498747 Rhodospirillum rubrum CODH(CooS) AAC45123 1498748 Rhodospirillum rubrum CooC AAC45124 1498749Rhodospirillum rubrum CooT AAC45125 1498750 Rhodospirillum rubrum CooJAAC45126 1498751 Rhodospirillum rubrum CODH-II (CooS-II) YP_35895778044574 Carboxydothermus hydrogenoformans CooF YP_358958 78045112Carboxydothermus hydrogenoformans

Anaerobic growth on synthesis gas and methanol in the absence of anexternal electron acceptor is conferred upon the host organism with MTRand ACS/CODH activity by allowing pyruvate synthesis via pyruvateferredoxin oxidoreductase (PFOR). This enzyme allows reversibleconversion of pyruvate into acetyl-CoA. The PFOR from Desulfovibrioafricanus has been cloned and expressed in E. coli resulting in anactive recombinant enzyme that was stable for several days in thepresence of oxygen (Pieulle et al., J. Bacteriol. 179.18:5684-5692(1997)). Oxygen stability is relatively uncommon in PFORs but can beconferred by a 60 residue extension in the polypeptide chain of the D.africanus enzyme. The M. thermoacetica PFOR is also well characterized(Menon and Ragsdale Biochemistry 36.28:8484-8494 (1997)) and was shownto have high activity in the direction of pyruvate synthesis duringautotrophic growth (Furdui and Ragsdale, J. Biol. Chem.275.37:28494-28499 (2000)). Further, E. coli possesses anuncharacterized open reading frame, ydbK, that encodes a protein that is51% identical to the M. thermoacetica PFOR. Evidence for pyruvateoxidoreductase activity in E. coli has been described (Blaschkowski etal., Eur. J. Biochem. 123.3:563-569 (1982)). The protein sequences ofthese exemplary PFOR enzymes can be identified by the following GenBankaccession numbers. Several additional PFOR enzymes are described in thefollowing review (Ragsdale, S. W., Chem. Rev. 103.6:2333-2346 (2003)):

Protein GenBank ID GI number Organism Por CAA70873.1 1770208Desulfovibrio africanus Por YP_428946.1 83588937 Moorella thermoaceticaYdbK NP_415896.1 16129339 Escherichia coli

The conversion of pyruvate into acetyl-CoA can be catalyzed by severalother enzymes or their combinations thereof. For example, pyruvatedehydrogenase can transform pyruvate into acetyl-CoA with theconcomitant reduction of a molecule of NAD into NADH. It is amulti-enzyme complex that catalyzes a series of partial reactions whichresults in acylating oxidative decarboxylation of pyruvate. The enzymecomprises of three subunits: the pyruvate decarboxylase (E1),dihydrolipoamide acyltransferase (E2) and dihydrolipoamide dehydrogenase(E3). This enzyme is naturally present in several organisms, includingE. coli and S. cerevisiae. In the E. coli enzyme, specific residues inthe E1 component are responsible for substrate specificity (Bisswanger,H., J. Biol. Chem. 256:815-82 (1981); Bremer, J., Eur. J. Biochem.8:535-540 (1969); Gong et al., J. Biol. Chem. 275:13645-13653 (2000)).Enzyme engineering efforts have improved the E. coli PDH enzyme activityunder anaerobic conditions (Kim et al., J. Bacteriol. 190:3851-3858(2008); Kim et al., Appl. Environ. Microbiol. 73:1766-1771 (2007); Zhouet al., Biotechnol. Lett. 30:335-342 (2008)). In contrast to the E. coliPDH, the B. subtilis complex is active and required for growth underanaerobic conditions (Nakano et al., J. Bacteriol. 179:6749-6755(1997)). The Klebsiella pneumoniae PDH, characterized during growth onglycerol, is also active under anaerobic conditions (Menzel et al., J.Biotechnol. 56:135-142 (1997)). Crystal structures of the enzyme complexfrom bovine kidney (Zhou et al., Proc. Natl. Acad. Sci. U.S.A.98:14802-14807 (2001)) and the E2 catalytic domain from Azotobactervinelandii are available (Mattevi et al., Science 255:1544-1550 (1992)).Yet another enzyme that can catalyze this conversion is pyruvate formatelyase. This enzyme catalyzes the conversion of pyruvate and CoA intoacetyl-CoA and formate. Pyruvate formate lyase is a common enzyme inprokaryotic organisms that is used to help modulate anaerobic redoxbalance. Exemplary enzymes can be found in Escherichia coli encoded bypflB (Knappe and Sawers, FEMS. Microbiol Rev. 6:383-398 (1990)),Lactococcus lactis (Melchiorsen et al., Appl Microbiol Biotechnol58:338-344 (2002)), and Streptococcus mutans (Takahashi-Abbe et al.,Oral. Microbiol Immunol. 18:293-297 (2003)). E. coli possesses anadditional pyruvate formate lyase, encoded by tdcE, that catalyzes theconversion of pyruvate or 2-oxobutanoate to acetyl-CoA or propionyl-CoA,respectively (Hesslinger et al., Mol. Microbiol 27:477-492 (1998)). BothpflB and tdcE from E. coli require the presence of pyruvate formatelyase activating enzyme, encoded by pflA. Further, a short proteinencoded by yfiD in E. coli can associate with and restore activity tooxygen-cleaved pyruvate formate lyase (Vey et al., Proc. Natl. Acad.Sci. U.S.A. 105:16137-16141 (2008). Note that pflA and pflB from E. coliwere expressed in S. cerevisiae as a means to increase cytosolicacetyl-CoA for butanol production as described in WO/2008/080124].Additional pyruvate formate lyase and activating enzyme candidates,encoded by pfl and act, respectively, are found in Clostridiumpasteurianum (Weidner et al., J Bacteriol. 178:2440-2444 (1996)).

Further, different enzymes can be used in combination to convertpyruvate into acetyl-CoA. For example, in S. cerevisiae, acetyl-CoA isobtained in the cytosol by first decarboxylating pyruvate to formacetaldehyde; the latter is oxidized to acetate by acetaldehydedehydrogenase and subsequently activated to form acetyl-CoA byacetyl-CoA synthetase. Acetyl-CoA synthetase is a native enzyme inseveral other organisms including E. coli (Kumari et al., J. Bacteriol.177:2878-2886 (1995)), Salmonella enterica (Starai et al., Microbiology151:3793-3801 (2005); Starai et al., J. Biol. Chem. 280:26200-26205(2005)), and Moorella thermoacetica (described already). Alternatively,acetate can be activated to form acetyl-CoA by acetate kinase andphosphotransacetylase. Acetate kinase first converts acetate intoacetyl-phosphate with the accompanying use of an ATP molecule.Acetyl-phosphate and CoA are next converted into acetyl-CoA with therelease of one phosphate by phosphotransacetylase. Both acetate kinaseand phosphotransacetylyase are well-studied enzymes in severalClostridia and Methanosarcina thermophila

Yet another way of converting pyruvate to acetyl-CoA is via pyruvateoxidase. Pyruvate oxidase converts pyruvate into acetate, usingubiquione as the electron acceptor. In E. coli, this activity is encodedby poxB. PoxB has similarity to pyruvate decarboxylase of S. cerevisiaeand Zymomonas mobilis. The enzyme has a thiamin pyrophosphate cofactor(Koland and Gennis, Biochemistry 21:4438-4442 (1982)); O'Brien et al.,Biochemistry 16:3105-3109 (1977); O'Brien and Gennis, J. Biol. Chem.255:3302-3307 (1980)) and a flavin adenine dinucleotide (FAD) cofactor.Acetate can then be converted into acetyl-CoA by either acetyl-CoAsynthetase or by acetate kinase and phosphotransacetylase, as describedearlier. Some of these enzymes can also catalyze the reverse reactionfrom acetyl-CoA to pyruvate.

Unlike the redox neutral conversion of CO and MeOH to acetyl-CoA oracetate, the production of more highly reduced products such as ethanol,butanol, isobutanol, isopropanol, 1,4-butanediol, 1,3-butanediol,succinic acid, fumaric acid, malic acid, 4-hydroxybutyric acid,3-hydroxypropionic acid, lactic acid, adipic acid, methacrylic acid, andacrylic acid at the highest possible yield from gaseous substratesrelies on the extraction of additional reducing equivalents from both COand H₂ (for example, see ethanol formation in FIG. 4). Specifically,reducing equivalents are obtained by the conversion of CO and water toCO₂ via carbon monoxide dehydrogenase or directly from the activity of ahydrogen-utilizing hydrogenase which transfers electrons from H₂ to anacceptor such as ferredoxin, flavodoxin, FAD⁺, NAD⁺, or NADP⁺.

Native to E. coli and other enteric bacteria are multiple genes encodingup to four hydrogenases (Sawers, G., Antonie van Leeuwenhoek 66.1:57-881994); Sawers et al., J. Bacteriol. 168.1:398-404 (1986); Sawers andBoxer, Eur. J. Biochem. 156.2:265-275 (1986); Sawers et al., J.Bacteriol. 164.3:1324-1331 (1985)). Given the multiplicity of enzymeactivities E. coli or another host organism can provide sufficienthydrogenase activity to split incoming molecular hydrogen and reduce thecorresponding acceptor. Among the endogenous hydrogen-lyase enzymes ofE. coli are hydrogenase 3, a membrane-bound enzyme complex usingferredoxin as an acceptor, and hydrogenase 4 that also uses a ferredoxinacceptor. Hydrogenase 3 and 4 are encoded by the hyc and hyf geneclusters, respectively. Hydrogenase activity in E. coli is alsodependent upon the expression of the hyp genes whose correspondingproteins are involved in the assembly of the hydrogenase complexes(Rangarajan et al. J. Bacteriol. 190.4:1447-1458 (2008); Jacobi et al.,Arch. Microbiol. 158.6:444-451 (1992)). The M. thermoaceticahydrogenases are suitable candidates should the production host lacksufficient endogenous hydrogenase activity. M. thermoacetica can growwith CO₂ as the exclusive carbon source indicating that reducingequivalents are extracted from H₂ to enable acetyl-CoA synthesis via theWood-Ljungdahl pathway (Drake, H. L., J. Bacteriol. 150.2:702-709(1982); Kellum and Drake, J. Bacteriol. 160.1:466-469 (1984); Drake andDaniel Res. Microbial. 155.10:869-883 (2004)) (see FIG. 2). M.thermoacetica has homologs to several hyp, hyc, and hyf genes from E.coli. These protein sequences encoded for by these genes can beidentified by the following GenBank accession numbers. In addition,several gene clusters encoding hydrogenase functionality are present inM. thermoacetica and their corresponding protein sequences are alsoprovided below:

Protein GenBank ID GI number Organism HypA NP_417206 16130633Escherichia coli HypB NP_417207 16130634 Escherichia coli HypC NP_41720816130635 Escherichia coli HypD NP_417209 16130636 Escherichia coli HypENP_417210 226524740 Escherichia coli HypF NP_417192 16130619 Escherichiacoli

Proteins in M. thermoacetica whose genes are homologous to the E. colihyp genes are shown below:

Protein GenBank ID GI number Organism Moth_2175 YP_431007 83590998Moorella thermoacetica Moth_2176 YP_431008 83590999 Moorellathermoacetica Moth_2177 YP_431009 83591000 Moorella thermoaceticaMoth_2178 YP_431010 83591001 Moorella thermoacetica Moth_2179 YP_43101183591002 Moorella thermoacetica Moth_2180 YP_431012 83591003 Moorellathermoacetica Moth_2181 YP_431013 83591004 Moorella thermoacetica

Hydrogenase 3 proteins are shown below:

Protein GenBank ID GI number Organism HycA NP_417205 16130632Escherichia coli HycB NP_417204 16130631 Escherichia coli HycC NP_41720316130630 Escherichia coli HycD NP_417202 16130629 Escherichia coli HycENP_417201 16130628 Escherichia coli HycF NP_417200 16130627 Escherichiacoli HycG NP_417199 16130626 Escherichia coli HycH NP_417198 16130625Escherichia coli HycI NP_417197 16130624 Escherichia coli

Hydrogenase 4 proteins are shown below:

Protein GenBank ID GI number Organism HyfA NP_416976 90111444Escherichia coli HyfB NP_416977 16130407 Escherichia coli HyfC NP_41697890111445 Escherichia coli HyfD NP_416979 16130409 Escherichia coli HyfENP_416980 16130410 Escherichia coli HyfF NP_416981 16130411 Escherichiacoli HyfG NP_416982 16130412 Escherichia coli HyfH NP_416983 16130413Escherichia coli HyfI NP_416984 16130414 Escherichia coli HyfJ NP_41698590111446 Escherichia coli HyfR NP_416986 90111447 Escherichia coli

Proteins in M. thermoacetica whose genes are homologous to the E. colihyc and/or hyf genes are shown below:

Protein GenBank ID GI number Organism Moth_2182 YP_431014 83591005Moorella thermoacetica Moth_2183 YP_431015 83591006 Moorellathermoacetica Moth_2184 YP_431016 83591007 Moorella thermoaceticaMoth_2185 YP_431017 83591008 Moorella thermoacetica Moth_2186 YP_43101883591009 Moorella thermoacetica Moth_2187 YP_431019 83591010 Moorellathermoacetica Moth_2188 YP_431020 83591011 Moorella thermoaceticaMoth_2189 YP_431021 83591012 Moorella thermoacetica Moth_2190 YP_43102283591013 Moorella thermoacetica Moth_2191 YP_431023 83591014 Moorellathermoacetica Moth_2192 YP_431024 83591015 Moorella thermoacetica

Additional hydrogenase-encoding gene clusters in M. thermoacetica areshown below:

Protein GenBank ID GI number Organism Moth_0439 YP_429313 83589304Moorella thermoacetica Moth_0440 YP_429314 83589305 Moorellathermoacetica Moth_0441 YP_429315 83589306 Moorella thermoaceticaMoth_0442 YP_429316 83589307 Moorella thermoacetica Moth_0809 YP_42967083589661 Moorella thermoacetica Moth_0810 YP_429671 83589662 Moorellathermoacetica Moth_0811 YP_429672 148283119 Moorella thermoaceticaMoth_0814 YP_429674 83589665 Moorella thermoacetica Moth_0815 YP_42967583589666 Moorella thermoacetica Moth_0816 YP_429676 83589667 Moorellathermoacetica Moth_1193 YP_430050 83590041 Moorella thermoaceticaMoth_1194 YP_430051 83590042 Moorella thermoacetica Moth_1195 YP_43005283590043 Moorella thermoacetica Moth_1196 YP_430053 83590044 Moorellathermoacetica Moth_1717 YP_430562 83590553 Moorella thermoaceticaMoth_1718 YP_430563 83590554 Moorella thermoacetica Moth_1719 YP_43056483590555 Moorella thermoacetica Moth_1883 YP_430726 83590717 Moorellathermoacetica Moth_1884 YP_430727 83590718 Moorella thermoaceticaMoth_1885 YP_430728 83590719 Moorella thermoacetica Moth_1886 YP_43072983590720 Moorella thermoacetica Moth_1887 YP_430730 83590721 Moorellathermoacetica Moth_1888 YP_430731 83590722 Moorella thermoaceticaMoth_1452 YP_430305 83590296 Moorella thermoacetica Moth_1453 YP_43030683590297 Moorella thermoacetica Moth_1454 YP_430307 83590298 Moorellathermoacetica

1,3-butanediol production can be achieved in recombinant E. coli byvarious alternate pathways described in FIG. 4 and FIG. 5. All pathwaysfirst convert two molecules of acetyl-CoA into one molecule ofacetoacetyl-CoA employing a thiolase.

Acetoacetyl-CoA thiolase converts two molecules of acetyl-CoA into onemolecule each of acetoacetyl-CoA and CoA. Exemplary acetoacetyl-CoAthiolase enzymes include the gene products of atoB from E. coli (Martinet al., Nat. Biotechnol. 21.7:796-802 (2003)), thlA and thlB from C.acetobutylicum (Hanai et al., Appl. Environ. Microbiol. 73.24:7814-7818(2007); Winzer et al. J. Mol. Microbiol. Biotechnol. 2.4:531-541(2000)), and ERG10 from S. cerevisiae (Hiser et al., J. Biol. Chem.269.50:31383-31389 (1994)). Information related to these proteins andgene can be found using the information below:

Protein GenBank ID GI number Organism AtoB NP_416728 16130161Escherichia coli ThlA NP_349476.1 15896127 Clostridium acetobutylicumThlB NP_149242.1 15004782 Clostridium acetobutylicum ERG10 NP_0152976325229 Saccharomyces cerevisiae

One pathway from acetoacetyl-CoA entails its reduction to3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase (ketone reducing).This can subsequently be converted to 3-hydroxybutyraldehyde via aCoA-dependent aldehyde reductase called 3-hydroxybutyryl-CoA reductase.3-hydroxybutyraldehyde can eventually be reduced to the product 1,3-BDOby 3-hydroxybutyraldehyde reductase. Alternatively, 3-hydroxybutyryl-CoAcan be reduced directly to 1,3-BDO by an alcohol-forming CoA-dependent3-hydroxybutyryl-CoA reductase. The genes for each of the steps in thepathway are described below.

Acetoacetyl-CoA reductase (ketone reducing) catalyzing the reduction ofacetoacetyl-CoA to 3-hydroxybutyryl-CoA participates in the acetyl-CoAfermentation pathway to butyrate in several species of Clostridia andhas been studied in detail (Jones and Woods, Microbiol. Rev.50.4:484-524 (1986)). The enzyme from Clostridium acetobutylicum,encoded by hbd, has been cloned and functionally expressed in E. coli(Youngleson et al., J. Bacteriol. 171.12:6800-6807 (1989)).Additionally, subunits of two fatty acid oxidation complexes in E. coli,encoded by fadB and fadJ, function as 3-hydroxyacyl-CoA dehydrogenases(Binstock and Schulz, Methods Enzymol. 71 Pt. C: 403-411 (1981)). Othergenes demonstrated to reduce acetoacetyl-CoA to 3-hydroxybutyryl-CoA arephbB from Zoogloea ramigera (Ploux et al., Eur. J. Biochem.171.1:177-182 (1988)) and phaB from Rhodobacter sphaeroides (Alber etal., Mol. Microbiol. 61.2:297-309 (2006)). The former gene isNADPH-dependent, its nucleotide sequence has been determined (Peoplesand Sinskey, Mol. Microbiol. 3.3:349-357 (1989)) and the gene has beenexpressed in E. coli. Substrate specificity studies on the gene led tothe conclusion that it could accept 3-oxopropionyl-CoA as a substratebesides acetoacetyl-CoA (Ploux et al., Eur. J. Biochem. 171.1:177-182(1988)). Additional genes include Hbd1 (C-terminal domain) and Hbd2(N-terminal domain) in Clostridium kluyveri (Hillmer and Gottschalk,Biochim. Biophys. Acta 3334:12-23 (1974)) and HSD17B10 in Bos taurus(Wakil et al., J. Biol. Chem. 207.2:631-638 (1954)). A summary of thegene and protein information are shown below:

Protein GENBANK ID GI NUMBER ORGANISM fadB P21177.2 119811 Escherichiacoli fadJ P77399.1 3334437 Escherichia coli Hbd2 EDK34807.1 146348271Clostridium kluyveri Hbd1 EDK32512.1 146345976 Clostridium kluyveri hbdP52041.2 Clostridium acetobutylicum HSD17B10 O02691.3 3183024 Bos TaurusphbB P23238.1 130017 Zoogloea ramigera phaB YP_353825.1 77464321Rhodobacter sphaeroides

A number of similar enzymes have been found in other species ofClostridia and in Metallosphaera sedula (Berg et al., Science318.5857:1782-1786 (2007)) as shown below:

Protein GenBank ID GI number Organism Hbd NP_349314.1 NP_349314.1Clostridium acetobutylicum Hbd AAM14586.1 AAM14586.1 Clostridiumbeijerinckii Msed_1423 YP_001191505 YP_001191505 Metallosphaera sedulaMsed_0399 YP_001190500 YP_001190500 Metallosphaera sedula Msed_0389YP_001190490 YP_001190490 Metallosphaera sedula Msed_1993 YP_001192057YP_001192057 Metallosphaera sedula

Several acyl-CoA dehydrogenases are capable of reducing an acyl-CoA toits corresponding aldehyde and can be used for catalyzing the3-hydroxybutyryl-CoA reductase (aldehyde forming) activity. Exemplarygenes that encode such enzymes include the Acinetobacter calcoaceticusacr1 encoding a fatty acyl-CoA reductase (Reiser and Somerville, J.Bacteriol. 179.9:2969-2975 (1997)), the Acinetobacter sp. M-1 fattyacyl-CoA reductase Ishige et al. Appl. Environ. Microbiol. 68.3:192-195(2002)), and a CoA- and NADP-dependent succinate semialdehydedehydrogenase encoded by the sucD gene in Clostridium kluyveri (Sohlingand Gottschalk, J. Bacteriol. 178.3:871-880 (1996); Sohling andGottschalk J. Bacteriol. 178.3:871-880 (1996)). SucD of P. gingivalis isanother succinate semialdehyde dehydrogenase (Takahashi et al., J.Bacteriol. 182.17:4704-4710 (2000)). The enzyme acylating acetaldehydedehydrogenase in Pseudomonas sp, encoded by bphG, is yet another enzymedemonstrated to oxidize and acylate acetaldehyde, propionaldehyde,butyraldehyde, isobutyraldehyde and formaldehyde (Powlowski et al., J.Bacteriol. 175.2:377-385 (1993)). In addition to reducing acetyl-CoA toethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides hasbeen shown to oxidize the branched chain compound isobutyraldehyde toisobutyryl-CoA (Kazahaya et al., J. Gen. Appl. Microbiol. 18:43-55(1972); Koo et al., Biotechnol. Lett. 27.7:505-510 (2005)).Butyraldehyde dehydrogenase catalyzes a similar reaction, conversion ofbutyryl-CoA to butyraldehyde, in solventogenic organisms such asClostridium saccharoperbutylacetonicum (Kosaka et al., Biosci.Biotechnol. Biochem. 71.1:58-68 (2007)). Information related to thesegenes and proteins are show below:

Protein GENBANK ID GI NUMBER ORGANISM acr1 YP_047869.1 50086359Acinetobacter calcoaceticus acr1 AAC45217 1684886 Acinetobacter baylyiacr1 BAB85476.1 18857901 Acinetobacter sp. Strain M-1 sucD P38947.1172046062 Clostridium kluyveri sucD NP_904963.1 34540484 Porphyromonasgingivalis bphG BAA03892.1 425213 Pseudomonas sp adhE AAV66076.155818563 Leuconostoc mesenteroides bld AAP42563.1 31075383 Clostridiumsaccharoperbutylacetonicum

An additional enzyme type that converts an acyl-CoA to its correspondingaldehyde is malonyl-CoA reductase which transforms malonyl-CoA tomalonic semialdehyde. Malonyl-CoA reductase is a key enzyme inautotrophic carbon fixation via the 3-hydroxypropionate cycle inthermoacidophilic archaeal bacteria (Berg et al., Science318.5857:1782-1786 (2007); Thauer, R. K., Science 318.5857:1732-1733).The enzyme utilizes NADPH as a cofactor and has been characterized inMetallosphaera and Sulfolobus spp (Alber et al., J. Bacteriol. 188.24:8551-8559 (2006); Hugler et al. 2404-10). The enzyme is encoded byMsed_(—)0709 in Metallosphaera sedula ((Alber et al., J. Bacteriol.188.24: 8551-8559 (2006); (Berg et al., Science 318.5857:1782-1786(2007)). A gene encoding a malonyl-CoA reductase from Sulfolobustokodaii was cloned and heterologously expressed in E. coli (Alber etal., J. Bacteriol. 188.24: 8551-8559 (2006); Alber et al., Mol.Microbiol. 61.2:297-309 (2006). This enzyme has also been shown tocatalyze the conversion of methylmalonyl-CoA to its correspondingaldehyde Although the aldehyde dehydrogenase functionality of theseenzymes is similar to the bifunctional dehydrogenase from Chloroflexusaurantiacus, there is little sequence similarity. Both malonyl-CoAreductase enzyme candidates have high sequence similarity toaspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reductionand concurrent dephosphorylation of aspartyl-4-phosphate to aspartatesemialdehyde. Additional genes can be found by sequence homology toproteins in other organisms including Sulfolobus solfataricus andSulfolobus acidocaldarius and have been listed below. Yet another genefor CoA-acylating aldehyde dehydrogenase is the ald gene fromClostridium beijerinckii (Toth et al., Appl. Environ. Microbiol.65.11:4973-4980 (1999)). This enzyme has been reported to reduceacetyl-CoA and butyryl-CoA to their corresponding aldehydes. This geneis very similar to eutE that encodes acetaldehyde dehydrogenase ofSalmonella typhimurium and E. coli (Toth et al., Appl. Environ.Microbiol. 65.11:4973-4980 (1999)). A summary of relevant gene andprotein information is shown below:

PROTEIN GENBANK ID GI NUMBER ORGANISM MSED_0709 YP_001190808.1 146303492Metallosphaera sedula mcr NP_378167.1 15922498 Sulfolobus tokodaii asd-2NP_343563.1 15898958 Sulfolobus solfataricus Saci_2370 YP_256941.170608071 Sulfolobus acidocaldarius Ald AAT66436 9473535 Clostridiumbeijerinckii eutE AAA80209 687645 Salmonella typhimurium eutE P774452498347 Escherichia col

Enzymes exhibiting 3-hydroxybutyraldehyde reductase activity (EC1.1.1.61) have been characterized in Ralstonia eutropha (Bravo et al.,J. Forensic Sci. 49.2:379-387 (2004)), Clostridium kluyveri (Wolff andKenealy, Protein Expr. Purif. 6.2:206-212 (1995)) and Arabidopsisthaliana (Breitkreuz et al., J. Biol. Chem. 278.42: 41552-41556 (2003)).Yet another gene is the alcohol dehydrogenase adhl from Geobacillusthermoglucosidasius (Jeon et al., J. Biotechnol. 135.2:127-133 (2008)).A summary of gene and protein information is shown below:

PROTEIN GENBANK ID GI NUMBER ORGANISM 4hbd YP_726053.1 113867564Ralstonia eutropha H16 4hbd L21902.1 146348486 Clostridium kluyveri DSM555 4hbd Q94B07 75249805 Arabidopsis thaliana adhI AAR91477.1 40795502Geobacillus thermoglucosidasius M10EXG

Another exemplary enzyme is 3-hydroxyisobutyrate dehydrogenase whichcatalyzes the reversible oxidation of 3-hydroxyisobutyrate tomethylmalonate semialdehyde. This enzyme participates in valine, leucineand isoleucine degradation and has been identified in bacteria,eukaryotes, and mammals. The enzyme encoded by P84067 from Thermusthermophilus HB8 has been structurally characterized (Lokanath et al.,J. Mol. Biol. 352.4:905-917 (2005)). The reversibility of the human3-hydroxyisobutyrate dehydrogenase was demonstrated usingisotopically-labeled substrate (Manning and Pollitt, Biochem. J.231.2:481-484 (1985)). Additional genes encoding this enzyme include3hidh in Homo sapiens (Hawes et al. Methods Enzymol. 324:218-228 (2000))and Oryctolagus cuniculus (Hawes et al. Methods Enzymol. 324:218-228(2000); Chowdhury et al., Biosci. Biotechnol. Biochem. 60.12:2043-2047(1996)), mmsb in Pseudomonas aeruginosa, and dhat in Pseudomonas putida(Aberhart and Hsu, J. Chem. Soc. (Perkin 1) 6:1404-1406; Chowdhury etal., Biosci. Biotechnol. Biochem. 60.12:2043-2047 (1996)); Chowdhury etal., Biosci. Biotechnol. Biochem. 67.2:438-441 (2003)). Informationrelated to these gene and proteins is shown below:

PROTEIN GENBANK ID GI NUMBER ORGANISM P84067 P84067 75345323 Thermusthermophilus mmsb P28811.1 127211 Pseudomonas aeruginosa dhat Q59477.12842618 Pseudomonas putida 3hidh P31937.2 12643395 Homo sapiens 3hidhP32185.1 416872 Oryctolagus cuniculus

Other exemplary genes encoding enzymes that catalyze the conversion ofan aldehyde to alcohol (i.e., alcohol dehydrogenase or equivalentlyaldehyde reductase) and can serve as candidates for3-hydroxybutyraldehyde reductase include alrA encoding a medium-chainalcohol dehydrogenase for C2-C14 (Tani et al., Appl. Environ. Microbiol.66.12:5231-5335 (2000)), ADH2 from Saccharomyces cerevisiae (Atsumi etal., Nature 451.7174:86-89 (2008)), yqhD from E. coli which haspreference for molecules longer than C3 (Sulzenbacher et al., J. Mol.Biol. 342.2:489-502 (2004)), and bdh I and bdh II from C. acetobutylicumwhich converts butyraldehyde into butanol (Walter et al., J. Bacteriol.174.22:7149; 7158 (1992)). The gene product of yqhD catalyzes thereduction of acetaldehyde, malondialdehyde, propionaldehyde,butyraldehyde, and acrolein using NADPH as the cofactor (Perez et al.,J. Biol. Chem. 283.12:7346-7353 (2008)). ADH1 from Zymomonas mobilis hasbeen demonstrated to have activity on a number of aldehydes includingformaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, and acrolein(Kinoshita et al., Appl. Microbiol. Biotchenol. 22:249-254 (1985)).

The protein sequences for each of these exemplary gene products, ifavailable, can be found using the following GenBank accession numbers:

Protein GENBANK ID GI NUMBER ORGANISM alrA BAB12273.1 9967138Acinetobacter sp. strain M-1 ADH2 NP_014032.1 6323961 Saccharomycescerevisiae yqhD NP_417484.1 16130909 Escherichia coli bdh I NP_349892.115896543 Clostridium acetobutylicum bdh II NP_349891.1 15896542Clostridium acetobutylicum adhA YP_162971.1 56552132 Zymomonas mobilis

The alcohol-forming 3-hydroxybutyryl-CoA reductase can be catalyzedby_exemplary two-step oxidoreductases that convert an acyl-CoA toalcohol. These include enzymes that transform substrates such asacetyl-CoA to ethanol (e.g., adhE from E. coli (Kessler et al., FEBSLett. 281.1-2:59-63 (1991)) and butyryl-CoA to butanol (e.g. adhE2 fromC. acetobutylicum (Fontaine et al., J. Bacteriol. 184.3:821-830 (2002)).In addition to reducing acetyl-CoA to ethanol, the enzyme encoded byadhE in Leuconostoc mesenteroides has been shown to oxidize the branchedchain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al., J.Gen. App. Microbiol. 18:43-55 (1972); Koo et al., Biotechnol. Lett.27.7:505-510 (2005)). Relevant information to these genes and proteinsare shown below:

Protein GENBANK ID GI NUMBER ORGANISM adhE NP_415757.1 16129202Escherichia coli adhE2 AAK09379.1 12958626 Clostridium acetobutylicumadhE AAV66076.1 55818563 Leuconostoc mesenteroides

Another exemplary enzyme can convert malonyl-CoA to 3-HP. AnNADPH-dependent enzyme with this activity has characterized inChloroflexus aurantiacus where it participates in the3-hydroxypropionate cycle (Hugler et al., J. Bacteriol. 184.9:2404-2410(2002); Strauss and Fuchs, Eur. J. Biochem. 215.3:633-643 (1993)). Thisenzyme, with a mass of 300 kDa, is highly substrate-specific and showslittle sequence similarity to other known oxidoreductases (Hugler etal., J. Bacteriol. 184.9:2404-2410 (2002)). No enzymes in otherorganisms have been shown to catalyze this specific reaction, howeverthere is bioinformatic evidence that other organisms can have similarpathways (Klatt et al., Environ. Microbiol. 9.8:2067-2078 (2007)).Enzyme candidates in other organisms including Roseiflexus castenholzii,Erythrobacter sp. NAP1 and marine gamma proteobacterium HTCC2080 can beinferred by sequence similarity. Information related to these genes andproteins is shown below:

Protein GENBANK ID GI NUMBER ORGANISM mcr AAS20429.1 42561982Chloroflexus aurantiacus Rcas_2929 YP_001433009.1 156742880 Roseiflexuscastenholzii NAP1_02720 ZP_01039179.1 85708113 Erythrobacter sp. NAP1MGP2080_00535 ZP_01626393.1 119504313 marine gamma proteo- bacteriumHTCC2080

Longer chain acyl-CoA molecules can be reduced by enzymes such as thejojoba (Simmondsia chinensis) FAR which encodes an alcohol-forming fattyacyl-CoA reductase. Its overexpression in E. coli resulted in FARactivity and the accumulation of fatty alcohol (Metz et al., PlantPhysiol. 122.3: 635-644 (2000)). Information related to FAR is shownbelow:

Protein GENBANK ID GI NUMBER ORGANISM FAR AAD38039.1 5020215 Simmondsiachinensis

A second alternate pathway from acetoacetyl-CoA to 1,3-butanediolproceeds via the reduction of acetoacetyl-CoA into 3-oxobutyraldehydevia the CoA-dependent aldehyde forming acetoacetyl-CoA reductase.3-oxobutyraldehyde is next reduced to 3-hydroxybutyraldehyde by3-oxobutyraldehyde reductase (ketone reducing), and eventually, thisintermediate is reduced to 1,3-butanediol by a 3-hydroxybutyraldehydereductase. The enzymes and genes encoding these enzymes for each ofthese steps are listed below.

Exemplary candidates for acetoacetyl-CoA reductase (CoA-dependent,aldehyde forming) that catalyzes the transformation of acetoacetyl-CoAinto 3-oxobutyraldehyde are the same as those described for3-hydroxybutyryl-CoA reductase (aldehyde forming) described hereinabove.

There exist several exemplary alcohol dehydrogenases that convert aketone to a hydroxyl functional group and can be used for catalyzing the3-oxobutyraldehyde reductase (ketone-reducing) activity. Two suchenzymes from E. coli are encoded by malate dehydrogenase (mdh) andlactate dehydrogenase (ldhA). In addition, lactate dehydrogenase fromRalstonia eutropha has been shown to demonstrate high activities onsubstrates of various chain lengths such as lactate, 2-oxobutyrate,2-oxopentanoate and 2-oxoglutarate (Steinbuchel and Schlegel, Eur. J.Biochem. 130.2:329-334 (1983)). Conversion of the oxo functionality tothe hydroxyl group can also be catalyzed by 2-ketol,3-butanediolreductase, an enzyme reported to be found in rat and in human placenta(Suda et al., Arch. Biochem. Biophys. 176.2:610-620 (1976); Suda et al.,Biochem. Biophys. Res. Commun. 342.2:586-591 (1977)). All of theseenzymes can be use as a 3-oxobutyraldehyde reductase. An additionalenzyme for this step is the mitochondrial 3-hydroxybutyratedehydrogenase (bdh) from the human heart which has been cloned andcharacterized (Marks et al., J. Biol. Chem. 267.22:15459-15463 (1992)).This enzyme is a dehydrogenase that operates on a 3-hydroxyacid. Anotherexemplary alcohol dehydrogenase that converts acetone to isopropanol aswas shown in C. beijerinckii (Ismaiel et al., J. Bacteriol.175.16:5097-5105 (1993)) and T. brockii (Lamed and Zeikus, Biochem. J.195.1:183-190 (1981); Peretz and Burstein, Biochemistry 28.16:6549-6555(1989)). Methyl ethyl ketone (MEK) reductase, or alternatively,2-butanol dehydrogenase, catalyzes the reduction of MEK to form2-butanol. Exemplary enzymes can be found in Rhodococcus ruber (Kosjeket al., Biotechnol. Bioeng. 86.1:55-62 (2004)) and Pyrococcus furiosus(van der Oost et al., Eur. J. Biochem. 268.10:3062-3068 (2001)).Information related to these proteins and genes is shown below:

Protein GENBANK ID GI NUMBER ORGANISM mdh AAC76268.1 1789632 Escherichiacoli ldhA NP_415898.1 16129341 Escherichia coli ldh YP_725182.1113866693 Ralstonia eutropha bdh AAA58352.1 177198 Homo sapiens adhAAA23199.2 60592974 Clostridium beijerinckii adh P14941.1 113443Thermoanaerobacter brockii sadh CAD36475 21615553 Rhodococcus ruber adhA3288810 AAC25556 Pyrococcus furiosus

Another pathway from acetoacetyl-CoA proceeds via its reduction to4-hydroxy,2-butanone by the CoA-dependent, alcohol formingacetoacetyl-CoA reductase. This intermediate is then reduced to1,3-butanediol by 4-hydroxybutanone reductase. 4-hydroxybutanone canalso be formed from 3-oxobutyraldehyde by an aldehyde reducing3-oxobutyraldehyde reductase. Acetoacetyl-CoA reductase (CoA-dependent,alcohol forming) can utilize the same enzymes as those for thealcohol-forming 3-hydroxybutyryl-CoA reductase.

4-hydroxybutanone reductase activity can be obtained from the same genesas those described for 3-oxobutyraldehyde reductase. Additionally, anumber of organisms can catalyze the reduction of 4-hydroxy,2-butanoneto 1,3-butanediol, including those belonging to the genus Bacillus,Brevibacterium, Candida, and Klebsiella among others, as described byMatsuyama et al., U.S. Pat. No. 5,413,922.

Exemplary genes encoding enzymes that catalyze the conversion of analdehyde to alcohol (i.e., alcohol dehydrogenase or equivalentlyaldehyde reductase) include alrA encoding a medium-chain alcoholdehydrogenase for C₂-C₁₄ (Tani et al., App. Environ. Microbiol.66.12:5231-5235 (2000)), ADH2 from Saccharomyces cerevisiae (Atsumi etal., Nature 451.7174:86-89 (2008)), yqhD from E. coli which haspreference for molecules longer than C3 (Sulzenbacher et al., J. Mol.Biol. 342.2:489-502 (2004)), and bdh I and bdh II from C. acetobutylicumwhich converts butyraldehyde into butanol (Walter et al., J. Bacteriol.174.22:7149-7158 (1992)). The gene product of yqhD catalyzes thereduction of acetaldehyde, malondialdehyde, propionaldehyde,butyraldehyde, and acrolein using NADPH as the cofactor (Perez et al.,J. Biol. Chem. 283.12:7346-7353 (2008)). All of these genes can providethe 3-oxobutyraldehyde reductase (aldehyde reducing) activity forconverting 3-oxobutyraldehyde into 4-hydroxybutanone. ADH1 fromZymomonas mobilis has been demonstrated to have activity on a number ofaldehydes including formaldehyde, acetaldehyde, propionaldehyde,butyraldehyde, and acrolein (Kinoshita et al., App. Microbiol.Biotechnol. 22:249-254 (1985)).

Where available, the protein sequences for each of these exemplary geneproducts, can be found using the following GenBank accession numbers:

Protein GENBANK ID GI NUMBER ORGANISM alrA BAB12273.1 9967138Acinetobacter sp. strain M-1 ADH2 NP_014032.1 6323961 Saccharomycescerevisiae yqhD NP_417484.1 16130909 Escherichia coli bdh I NP_349892.115896543 Clostridium acetobutylicum bdh II NP_349891.1 15896542Clostridium acetobutylicum adhA YP_162971.1 56552132 Zymomonas mobilis

The conversion of acetoacetyl-CoA to acetoacetate can be carried out bya acetoacetyl-CoA transferase which conserves the energy stored in theCoA-ester bond. Several exemplary transferase enzymes capable ofcatalyzing this transformation are provided below. These enzymes eithernaturally exhibit the desired acetoacetyl-CoA transferase activity orthey can be engineered via directed evolution to acceptacetetoacetyl-CoA as a substrate with increased efficiency. Suchenzymes, either naturally or following directed evolution, are alsosuitable for catalyzing the conversion of 3-hydroxybutyryl-CoA to3-hydroxybutyrate via a transferase mechanism.

Acetoacetyl-CoA:acetyl-CoA transferase naturally convertsacetoacetyl-CoA and acetate to acetoacetate and acetyl-CoA. This enzymecan also accept 3-hydroxybutyryl-CoA as a substrate or could beengineered to do so. Exemplary enzymes include the gene products ofatoAD from E. coli (Hanai et al., Appl Environ Microbiol 73:7814-7818(2007)), ctfAB from C. acetobutylicum (Jojima et al., Appl MicrobiolBiotechnol 77:1219-1224 (2008)), and ctfAB from Clostridiumsaccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem.71:58-68 (2007)). Information related to these proteins and genes isshown below:

Protein GENBANK ID GI NUMBER ORGANISM AtoA P76459.1 2492994 Escherichiacoli AtoD P76458.1 2492990 Escherichia coli CtfA NP_149326.1 15004866Clostridium acetobutylicum CtfB NP_149327.1 15004867 Clostridiumacetobutylicum CtfA AAP42564.1 31075384 Clostridiumsaccharoperbutylacetonicum CtfB AAP42565.1 31075385 Clostridiumsaccharoperbutylacetonicum

Succinyl-CoA:3-ketoacid-CoA transferase naturally converts succinate tosuccinyl-CoA while converting a 3-ketoacyl-CoA to a 3-ketoacid.Exemplary succinyl-CoA:3:ketoacid-CoA transferases are present inHelicobacter pylori (Corthesy-Theulaz et al., J. Biol. Chem.272:25659-25667 (1997)), Bacillus subtilis (Stols et al., Protein. Expr.Purif. 53:396-403 (2007)), and Homo sapiens (Fukao et al., Genomics68:144-151 (2000); Tanaka et al., Mol. Hum. Reprod. 8:16-23 (2002)).Information related to these proteins and genes is shown below:

Protein GENBANK ID GI NUMBER ORGANISM HPAG1_0676 YP_627417 108563101Helicobacter pylori HPAG1_0677 YP_627418 108563102 Helicobacter pyloriScoA NP_391778 16080950 Bacillus subtilis ScoB NP_391777 16080949Bacillus subtilis OXCT1 NP_000427 4557817 Homo sapiens OXCT2 NP_07140311545841 Homo sapiens

Additional suitable acetoacetyl-CoA and 3-hydroxybutyryl-CoAtransferases are encoded by the gene products of cat1, cat2, and cat3 ofClostridium kluyveri. These enzymes have been shown to exhibitsuccinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA acetyltransferaseactivity, respectively (Seedorf et al., Proc. Natl. Acad. Sci. USA105:2128-2133 (2008); Sohling and Gottschalk, J Bacteriol 178:871-880(1996)). Similar CoA transferase activities are also present inTrichomonas vaginalis (van Grinsven et al., J. Biol. Chem. 283:1411-1418(2008)) and Trypanosoma brucei (Riviere et al., J. Biol. Chem.279:45337-45346 (2004)). Yet another transferase capable of the desiredconversions is butyryl-CoA:acetoacetate CoA-transferase. Exemplaryenzymes can be found in Fusobacterium nucleatum (Barker et al., J.Bacteriol. 152(1):201-7 (1982)), Clostridium SB4 (Barker et al., J.Biol. Chem. 253(4):1219-25 (1978)), and Clostridium acetobutylicum(Wiesenborn et al., Appl. Environ. Microbiol. 55(2):323-9 (1989)).Although specific gene sequences have not been indicated forbutyryl-CoA:acetoacetate CoA-transferase, the genes FN0272 and FN0273have been annotated as a butyrate-acetoacetate CoA-transferase (Kapatralet al., J. Bact. 184(7) 2005-2018 (2002)). Homologs in Fusobacteriumnucleatum such as FN1857 and FN1856 can have the desired acetoacetyl-CoAtransferase activity. FN1857 and FN1856 are located adjacent to manyother genes involved in lysine fermentation and are thus very can encodean acetoacetate:butyrate CoA transferase (Kreimeyer, et al., J. Biol.Chem. 282 (10) 7191-7197 (2007)). Additional genes/gene products fromPorphyrmonas gingivalis and Thermoanaerobacter tengcongensis can beidentified in a similar fashion (Kreimeyer, et al., J. Biol. Chem. 282(10) 7191-7197 (2007)). Information related to these proteins and genesis shown below:

Protein GENBANK ID GI NUMBER ORGANISM Cat1 P38946.1 729048 Clostridiumkluyveri Cat2 P38942.2 1705614 Clostridium kluyveri Cat3 EDK35586.1146349050 Clostridium kluyveri TVAG_395550 XP_001330176 123975034Trichomonas vaginalis G3 Tb11.02.0290 XP_828352 71754875 Trypanosomabrucei FN0272 NP_603179.1 19703617 Fusobacterium nucleatum FN0273NP_603180.1 19703618 Fusobacterium nucleatum FN1857 NP_602657.1 19705162Fusobacterium nucleatum FN1856 NP_602656.1 19705161 Fusobacteriumnucleatum PG1066 NP_905281.1 34540802 Porphyromonas gingivalis W83PG1075 NP_905290.1 34540811 Porphyromonas gingivalis W83 TTE0720NP_622378.1 20807207 Thermoanaerobacter tengcongensis MB4 TTE0721NP_622379.1 20807208 Thermoanaerobacter tengcongensis MB4

Acetoacetyl-CoA can be hydrolyzed to acetoacetate by acetoacetyl-CoAhydrolase. Similarly, 3-hydroxybutyryl-CoA can be hydrolyzed to3-hydroxybutyate by 3-hydroxybutyryl-CoA hydrolase. Many CoA hydrolases(EC 3.1.2.1) have broad substrate specificity and are suitable enzymesfor these transformations either naturally or following enzymeengineering. Though the sequences were not reported, severalacetoacetyl-CoA hydrolases were identified in the cytosol andmitochondrion of the rat liver (Aragon and Lowenstein, J. Biol. Chem.258(8):4725-4733 (1983)). Additionally, an enzyme from Rattus norvegicusbrain (Robinson et al., Biochem. Biophys. Res. Commun. 71:959-965(1976)) can react with butyryl-CoA, hexanoyl-CoA and malonyl-CoA. Theacot12 enzyme from the rat liver was shown to hydrolyze C2 to C6acyl-CoA molecules (Suematsu et al., Eur. J. Biochem. 268:2700-2709(2001)). Though its sequence has not been reported, the enzyme from themitochondrion of the pea leaf showed activity on acetyl-CoA,propionyl-CoA, butyryl-CoA, palmitoyl-CoA, oleoyl-CoA, succinyl-CoA, andcrotonyl-CoA (Zeiher and Randall, Plant. Physiol. 94:20-27 (1990)).Additionally, a glutaconate CoA-transferase from Acidaminococcusfermentans was transformed by site-directed mutagenesis into an acyl-CoAhydrolase with activity on glutaryl-CoA, acetyl-CoA and 3-butenoyl-CoA(Mack and Buckel, FEBS Lett. 405:209-212 (1997)). This indicates thatthe enzymes encoding succinyl-CoA:3-ketoacid-CoA transferases andacetoacetyl-CoA:acetyl-CoA transferases can also be used as hydrolaseswith certain mutations to change their function. The acetyl-CoAhydrolase, ACH1, from S. cerevisiae represents another candidatehydrolase (Buu et al., J. Biol. Chem. 278:17203-17209 (2003)).Information related to these proteins and genes is shown below:

Protein GENBANK ID GI NUMBER ORGANISM Acot12 NP_570103.1 18543355 Rattusnorvegicus GctA CAA57199 559392 Acidaminococcus fermentans GctB CAA57200559393 Acidaminococcus fermentans ACH1 NP_009538 6319456 Saccharomycescerevisiae

Another hydrolase enzyme is the human dicarboxylic acid thioesterase,acot8, which exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA,sebacyl-CoA, and dodecanedioyl-CoA (Westin et al., J. Biol. Chem.280:38125-38132 (2005)) and the closest E. coli homolog, tesB, which canalso hydrolyze a broad range of CoA thioesters (Naggert et al., J. Biol.Chem. 266:11044-11050 (1991)) including 3-hydroxybutyryl-CoA (Tseng etal., Appl. Environ. Microbiol. 75(10):3137-3145 (2009)). A similarenzyme has also been characterized in the rat liver (Deana, Biochem.Int. 26:767-773 (1992)). Other E. coli thioester hydrolases include thegene products of tesA (Bonner and Bloch, J. Biol. Chem. 247:3123-3133(1972)), ybgC (Kuznetsova et al., FEMS Microbiol. Rev. 29:263-279(2005); Zhuang et al., FEBS Lett. 516:161-163 (2002)), paal (Song etal., J. Biol. Chem. 281:11028-11038 (2006)), and ybdB (Leduc et al., J.Bacteriol. 189:7112-7126 (2007)). Information related to these proteinsand genes is shown below:

Protein GENBANK ID GI NUMBER ORGANISM Acot8 CAA15502 3191970 Homosapiens TesB NP_414986 16128437 Escherichia coli Acot8 NP_57011251036669 Rattus norvegicus TesA NP_415027 16128478 Escherichia coli YbgCNP_415264 16128711 Escherichia coli PaaI NP_415914 16129357 Escherichiacoli YbdB NP_415129 16128580 Escherichia coli

Additional hydrolase enzymes include 3-hydroxyisobutyryl-CoA hydrolasewhich has been described to efficiently catalyze the conversion of3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate during valinedegradation (Shimomura et al., J. Biol. Chem. 269:14248-14253 (1994)).Genes encoding this enzyme include hibch of Rattus norvegicus (Shimomuraet al., supra (1994); Shimomura et al., Methods Enzymol. 324:229-240(2000)) and Homo sapiens (Shimomura et al., supra (1994). Candidategenes by sequence homology include hibch of Saccharomyces cerevisiae andBC_(—)2292 of Bacillus cereus. BC_(—)2292 was shown to demonstrate3-hydroxybutyryl-CoA hydrolase activity and function as part of apathway for 3-hydroxybutyrate synthesis when engineered into Escherichiacoli (Lee et al., Appl. Microbiol. Biotechnol. 79:633-641 (2008)).Information related to these proteins and genes is shown below:

Protein GENBANK ID GI NUMBER ORGANISM Hibch Q5XIE6.2 146324906 Rattusnorvegicus Hibch Q6NVY1.2 146324905 Homo sapiens Hibch P28817.2 2506374Saccharomyces cerevisiae BC_2292 AP09256 29895975 Bacillus cereus ATCC14579

An alternative method for removing the CoA moiety from acetoacetyl-CoAor 3-hydroxybutyryl-CoA is to apply a pair of enzymes such as aphosphate-transferring acyltransferase and a kinase to impartacetoacetyl-CoA or 3-hydroxybutyryl-CoA synthetase activity. As usedherein, the combination of a phosphotransacylase and a kinase enzyme isreferred to as a “synthetase.” This activity allows the net hydrolysisof the CoA-ester of either molecule with the simultaneous generation ofATP. For example, the butyrate kinase (buk)/phosphotransbutyrylase (ptb)system from Clostridium acetobutylicum has been successfully applied toremove the CoA group from 3-hydroxybutyryl-CoA when functioning as partof a pathway for 3-hydroxybutyrate synthesis (Tseng et al., Appl.Environ. Microbiol. 75(10):3137-3145 (2009)). Specifically, the ptb genefrom C. acetobutylicum encodes an enzyme that can convert an acyl-CoAinto an acyl-phosphate (Walter et al. Gene 134(1): p. 107-11 (1993));Huang et al. J Mol Microbiol Biotechnol 2(1): p. 33-38 (2000).Additional ptb genes can be found in butyrate-producing bacterium L2-50(Louis et al. J. Bacteriol. 186:2099-2106 (2004)) and Bacillusmegaterium (Vazquez et al. Curr. Microbiol 42:345-349 (2001)).Additional exemplary phosphate-transferring acyltransferases includephosphotransacetylase, encoded by pta. The pta gene from E. coli encodesan enzyme that can convert acetyl-CoA into acetyl-phosphate, and viceversa (Suzuki, T. Biochim. Biophys. Acta 191:559-569 (1969)). Thisenzyme can also utilize propionyl-CoA instead of acetyl-CoA formingpropionate in the process (Hesslinger et al. Mol. Microbiol 27:477-492(1998)). Information related to these proteins and genes is shown below:

Protein GENBANK ID GI NUMBER ORGANISM Pta NP_416800.1 16130232Escherichia coli Ptb NP_349676 15896327 Clostridium acetobutylicum PtbAAR19757.1 38425288 butyrate-producing bacterium L2-50 Ptb CAC07932.110046659 Bacillus megaterium

Exemplary kinases include the E. coli acetate kinase, encoded by ackA(Skarstedt and Silverstein J. Biol. Chem. 251:6775-6783 (1976)), the C.acetobutylicum butyrate kinases, encoded by buk1 and buk2 ((Walter etal. Gene 134(1):107-111 (1993); Huang et al. J Mol Microbiol Biotechnol2(1):33-38 (2000)), and the E. coli gamma-glutamyl kinase, encoded byproB (Smith et al. J. Bacteriol. 157:545-551 (1984)). These enzymesphosphorylate acetate, butyrate, and glutamate, respectively. The ackAgene product from E. coli also phosphorylates propionate (Hesslinger etal. Mol. Microbiol 27:477-492 (1998)). Information related to theseproteins and genes is shown below:

Protein GENBANK ID GI NUMBER ORGANISM AckA NP_416799.1 16130231Escherichia coli Buk1 NP_349675 15896326 Clostridium acetobutylicum Buk2Q97II1 20137415 Clostridium acetobutylicum ProB NP_414777.1 16128228Escherichia coli

The hydrolysis of acetoacetyl-CoA or 3-hydroxybutyryl-CoA canalternatively be carried out by a single enzyme or enzyme complex thatexhibits acetoacetyl-CoA or 3-hydroxybutyryl-CoA synthetase activity.This activity enables the net hydrolysis of the CoA-ester of eithermolecule, and in some cases, results in the simultaneous generation ofATP. For example, the product of the LSC1 and LSC2 genes of S.cerevisiae and the sucC and sucD genes of E. coli naturally form asuccinyl-CoA synthetase complex that catalyzes the formation ofsuccinyl-CoA from succinate with the concomitant consumption of one ATP,a reaction which is reversible in vivo (Gruys et al., U.S. Pat. No.5,958,745, filed Sep. 28, 1999). Information related to these proteinsand genes is shown below:

Protein GENBANK ID GI NUMBER ORGANISM SucC NP_415256.1 16128703Escherichia coli SucD AAC73823.1 1786949 Escherichia coli LSC1 NP_0147856324716 Saccharomyces cerevisiae LSC2 NP_011760 6321683 Saccharomycescerevisiae

Additional exemplary CoA-ligases include the rat dicarboxylate-CoAligase for which the sequence is yet uncharacterized (Vamecq et al.,Biochemical J. 230:683-693 (1985)), either of the two characterizedphenylacetate-CoA ligases from P. chrysogenum (Lamas-Maceiras et al.,Biochem. J. 395:147-155 (2005); Wang et al., Biochem Biophy Res Commun360(2):453-458 (2007)), the phenylacetate-CoA ligase from Pseudomonasputida (Martinez-Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)),and the 6-carboxyhexanoate-CoA ligase from Bacilis subtilis (Bower etal., J. Bacteriol. 178(14):4122-4130 (1996)). Additional candidateenzymes are acetoacetyl-CoA synthetases from Mus musculus (Hasegawa etal., Biochim. Biophys. Acta 1779:414-419 (2008)) and Homo sapiens(Ohgami et al., Biochem. Pharmacol. 65:989-994 (2003)), which naturallycatalyze the ATP-dependant conversion of acetoacetate intoacetoacetyl-CoA. 4-Hydroxybutyryl-CoA synthetase activity has beendemonstrated in Metallosphaera sedula (Berg et al., Science318:1782-1786 (2007)). This function has been assigned to theMsed_(—)1422 gene. Information related to these proteins and genes isshown below:

GI Protein GENBANK ID NUMBER ORGANISM Phl CAJ15517.1 77019264Penicillium chrysogenum PhlB ABS19624.1 152002983 Penicilliumchrysogenum PaaF AAC24333.2 22711873 Pseudomonas putida BioW NP_390902.250812281 Bacillus subtilis AACS NP_084486.1 21313520 Mus musculus AACSNP_076417.2 31982927 Homo sapiens Msed_1422 YP_001191504 146304188Metallosphaera sedula

ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is another enzymethat couples the conversion of acyl-CoA esters to their correspondingacids with the concurrent synthesis of ATP. Several enzymes with broadsubstrate specificities have been described in the literature. ACD Ifrom Archaeoglobus fulgidus, encoded by AF1211, was shown to operate ona variety of linear and branched-chain substrates including acetyl-CoA,propionyl-CoA, butyryl-CoA, acetate, propionate, butyrate, isobutyryate,isovalerate, succinate, fumarate, phenylacetate, indoleacetate (Musfeldtet al., J. Bacteriol. 184:636-644 (2002)). The enzyme from Haloarculamarismortui (annotated as a succinyl-CoA synthetase) accepts propionate,butyrate, and branched-chain acids (isovalerate and isobutyrate) assubstrates, and was shown to operate in the forward and reversedirections (Brasen et al., Arch. Microbiol. 182:277-287 (2004)). The ACDencoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculumaerophilum showed the broadest substrate range of all characterizedACDs, reacting with acetyl-CoA, isobutyryl-CoA (preferred substrate) andphenylacetyl-CoA (Brasen et al., supra (2004)). The enzymes from A.fulgidus, H. marismortui and P. aerophilum have all been cloned,functionally expressed, and characterized in E. coli (Musfeldt et al.,supra; Brasen et al., supra (2004)). Information related to theseproteins and genes is shown below:

Protein GENBANK ID GI NUMBER ORGANISM AF1211 NP_070039.1 11498810Archaeoglobus fulgidus DSM 4304 scs YP_135572.1 55377722 Haloarculamarismortui ATCC 43049 PAE3250 NP_560604.1 18313937 Pyrobaculumaerophilum str. IM2

The conversion of 3-hydroxybutyrate to 3-hydroxybutyraldehyde can becarried out by a 3-hydroxybutyrate reductase. Similarly, the conversionof acetoacetate to acetoacetaldehyde can be carried out by anacetoacetate reductase. A suitable enzyme for these transformations isthe aryl-aldehyde dehydrogenase, or equivalently a carboxylic acidreductase, from Nocardia iowensis. Carboxylic acid reductase catalyzesthe magnesium, ATP and NADPH-dependent reduction of carboxylic acids totheir corresponding aldehydes (Venkitasubramanian et al., J. Biol. Chem.282:478-485 (2007)). This enzyme, encoded by car, was cloned andfunctionally expressed in E. coli (Venkitasubramanian et al., J. Biol.Chem. 282:478-485 (2007)). Expression of the npt gene product improvedactivity of the enzyme via post-transcriptional modification. The nptgene encodes a specific phosphopantetheine transferase (PPTase) thatconverts the inactive apo-enzyme to the active holo-enzyme. The naturalsubstrate of this enzyme is vanillic acid, and the enzyme exhibits broadacceptance of aromatic and aliphatic substrates (Venkitasubramanian etal., in Biocatalysis in the Pharmaceutical and Biotechnology Industires,ed. R. N. Patel, Chapter 15, pp. 425-440, CRC Press LLC, Boca Raton,Fla. (2006)). Information related to these proteins and genes is shownbelow:

Protein GI NUMBER GENBANK ID ORGANISM Car 40796035 AAR91681.1 Nocardiaiowensis (sp. NRRL 5646) Npt 114848891 ABI83656.1 Nocardia iowensis (sp.NRRL 5646)

Additional car and npt genes can be identified based on sequencehomology.

GI Protein GENBANK ID NUMBER ORGANISM fadD9 YP_978699.1 121638475Mycobacterium bovis BCG BCG_2812c YP_978898.1 121638674 Mycobacteriumbovis BCG nfa20150 YP_118225.1 54023983 Nocardia farcinica IFM 10152nfa40540 YP_120266.1 54026024 Nocardia farcinica IFM 10152 SGR_6790YP_001828302.1 182440583 Streptomyces griseus subsp. griseus NBRC 13350SGR_665 YP_001822177.1 182434458 Streptomyces griseus subsp. griseusNBRC 13350 MSMEG_2956 YP_887275.1 118473501 Mycobacterium smegmatis MC2155 MSMEG_5739 YP_889972.1 118469671 Mycobacterium smegmatis MC2 155MSMEG_2648 YP_886985.1 118471293 Mycobacterium smegmatis MC2 155MAP1040c NP_959974.1 41407138 Mycobacterium avium subsp.paratuberculosis K-10 MAP2899c NP_961833.1 41408997 Mycobacterium aviumsubsp. paratuberculosis K-10 MMAR_2117 YP_001850422.1 183982131Mycobacterium marinum M MMAR_2936 YP_001851230.1 183982939 Mycobacteriummarinum M MMAR_1916 YP_001850220.1 183981929 Mycobacterium marinum MTpauDRAFT_33060 ZP_04027864.1 227980601 Tsukamurella paurometabola DSM20162 TpauDRAFT_20920 ZP_04026660.1 227979396 Tsukamurella paurometabolaDSM 20162 CPCC7001_1320 ZP_05045132.1 254431429 Cyanobium PCC7001DDBDRAFT_0187729 XP_636931.1 66806417 Dictyostelium discoideum AX4

An additional enzyme candidate found in Streptomyces griseus is encodedby the griC and griD genes. This enzyme is believed to convert3-amino-4-hydroxybenzoic acid to 3-amino-4-hydroxybenzaldehyde asdeletion of either griC or griD led to accumulation of extracellular3-acetylamino-4-hydroxybenzoic acid, a shunt product of3-amino-4-hydroxybenzoic acid metabolism (Suzuki, et al., J. Antibiot.60(6):380-387 (2007)). Co-expression of griC and griD with SGR_(—)665,an enzyme similar in sequence to the Nocardia iowensis npt, can bebeneficial. Information related to these proteins and genes is shownbelow:

GI Protein NUMBER GENBANK ID ORGANISM griC 182438036 YP_001825755.1Streptomyces griseus subsp. griseus NBRC 13350 griD 182438037YP_001825756.1 Streptomyces griseus subsp. griseus NBRC 13350

An enzyme with similar characteristics, alpha-aminoadipate reductase(AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in somefungal species. This enzyme naturally reduces alpha-aminoadipate toalpha-aminoadipate semialdehyde. The carboxyl group is first activatedthrough the ATP-dependent formation of an adenylate that is then reducedby NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizesmagnesium and requires activation by a PPTase. Enzyme candidates for AARand its corresponding PPTase are found in Saccharomyces cerevisiae(Morris et al., Gene 98:141-145 (1991)), Candida albicans (Guo et al.,Mol. Genet. Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe(Ford et al., Curr. Genet. 28:131-137 (1995)). The AAR from S. pombeexhibited significant activity when expressed in E. coli (Guo et al.,Yeast 21:1279-1288 (2004)). The AAR from Penicillium chrysogenum acceptsS-carboxymethyl-L-cysteine as an alternate substrate, but did not reactwith adipate, L-glutamate or diaminopimelate (Hijarrubia et al., J.Biol. Chem. 278:8250-8256 (2003)). The gene encoding the P. chrysogenumPPTase has not been identified to date. Information related to theseproteins and genes is shown below:

Protein GI NUMBER GENBANK ID ORGANISM LYS2 171867 AAA34747.1Saccharomyces cerevisiae LYS5 1708896 P50113.1 Saccharomyces cerevisiaeLYS2 2853226 AAC02241.1 Candida albicans LYS5 28136195 AAO26020.1Candida albicans Lys1p 13124791 P40976.3 Schizosaccharomyces pombe Lys7p1723561 Q10474.1 Schizosaccharomyces pombe Lys2 3282044 CAA74300.1Penicillium chrysogenum

Essentially any of these CAR or CAR-like enzymes can exhibit3-hydroxybutyrate or acetoacetate reductase activity or can beengineered to do so.

The requisite 3-hydroxybutyrate dehydrogenase catalyzes the reduction ofacetoacetate to form 3-hydroxybutyrate. Exemplary enzymes can be foundin Rhodococcus ruber (Kosjek et al., Biotechnol Bioeng. 86:55-62 (2004))and Pyrococcus furiosus (van der et al., Eur. J. Biochem. 268:3062-3068(2001)). Additional secondary alcohol dehydrogenase enzymes capable ofthis transformation include adh from C. beijerinckii (Hanai et al., ApplEnviron Microbiol 73:7814-7818 (2007); Jojima et al., Appl MicrobiolBiotechnol 77:1219-1224 (2008)) and adh from Thermoanaerobacter brockii(Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007); Peretz etal., Anaerobe 3:259-270 (1997)). The cloning of the bdhA gene fromRhizobium (Sinorhizobium) Meliloti into E. coli conferred the ability toutilize 3-hydroxybutyrate as a carbon source (Aneja and Charles, J.Bacteriol. 181(3):849-857 (1999)). Additional 3-hydroxybutyratedehydrogenase can be found in Pseudomonas fragi (Ito et al., J. Mol.Biol. 355(4) 722-733 (2006)) and Ralstonia pickettii (Takanashi et al.,Antonie van Leeuwenoek, 95(3):249-262 (2009)). Information related tothese proteins and genes is shown below:

Protein GENBANK ID GI NUMBER ORGANISM Sadh CAD36475 21615553 Rhodococcusrubber AdhA AAC25556 3288810 Pyrococcus furiosus Adh P14941.1 113443Thermoanaerobobacter brockii Adh AAA23199.2 60592974 Clostridiumbeijerinckii BdhA NP_437676.1 16264884 Rhizobium (Sinorhizobium)Meliloti PRK13394 BAD86668.1 57506672 Pseudomonas fragi Bdh1 BAE72684.184570594 Ralstonia pickettii Bdh2 BAE72685.1 84570596 Ralstoniapickettii Bdh3 BAF91602.1 158937170 Ralstonia pickettii

Engineering the capability to convert CO₂, CO, and/or H₂ intoacetyl-CoA, the central metabolite from which all cell mass componentsand many valuable products can be derived, into a foreign host such asE. coli can be accomplished following the expression of exogenous genesthat encode various proteins of the Wood-Ljungdahl pathway. This pathwayis highly active in acetogenic organisms such as Moorella thermoacetica(formerly, Clostridium thermoaceticum), which has been the modelorganism for elucidating the Wood-Ljungdahl pathway since its isolationin 1942 (Fontaine et al., J. Bacteriol. 43.6:701-715 (1942)). TheWood-Ljungdahl pathway comprises of two branches: the Eastern (ormethyl) branch that enables the conversion of CO₂ tomethyltetrahydrofolate (Me-THF) and the Western (or carbonyl) branchthat enables the conversion of methyl-THF, CO, and Coenzyme-A intoacetyl-CoA (FIG. 5). Herein we describe a non-naturally occurringmicroorganism expressing genes encoding enzymes that catalyze the methyland carbonyl branches of the Wood-Ljungdahl pathway. Such an organism iscapable of converting CO, CO₂, and/or H₂ into acetyl-CoA, cell mass, andproducts.

In some embodiments, a non-naturally occurring organism utilizingpathways shown in FIG. 5 exhibit three capabilities: 1) a functionalmethyl branch of the Wood-Ljungdahl pathway which enables the conversionof THF and CO₂ to 5-methyl-tetrahydrofolate, 2) the ability to combineCO, Coenzyme A, and the methyl group of Me-THF to form acetyl-CoA, and3) the ability to synthesize 1,3-butanediol from acetyl-CoA.

Such non-naturally occurring organisms are able to ‘fix’ carbon fromexogenous CO and/or exogenous or endogenously produced CO₂ to synthesizeacetyl-CoA, cell mass, and products. A host organism engineered withthese capabilities that also naturally possesses the capability foranaplerosis (e.g., E. coli) can grow on the syngas-generated acetyl-CoAin the presence of a suitable external electron acceptor such asnitrate. This electron acceptor is required to accept electrons from thereduced quinone formed via succinate dehydrogenase. A further advantageof adding an external electron acceptor is that additional energy forcell growth, maintenance, and product formation can be generated fromrespiration of acetyl-CoA. An alternative strategy involves engineeringa pyruvate ferredoxin oxidoreductase (PFOR) enzyme or other enzymes thatconvert pyruvate into acetyl-CoA into the strain to enable synthesis ofbiomass precursors in the absence of an external electron acceptor. Afurther characteristic of the engineered organism is the capability forextracting reducing equivalents from molecular hydrogen. This enables ahigh yield of reduced products such as ethanol, butanol, isobutanol,isopropanol, 1,4-butanediol, 1,3-butanediol, succinic acid, fumaricacid, malic acid, 4-hydroxybutyric acid, 3-hydroxypropionic acid, lacticacid, adipic acid, methacrylic acid, and acrylic acid.

A non-naturally occurring organism can produce acetyl-CoA, cell mass,and targeted chemicals, more specifically 1,3-butanediol, from: 1) CO,2) CO₂ and H₂, 3) CO, CO₂, and H₂, 4) synthesis gas comprising CO andH₂, 5) synthesis gas comprising CO, CO₂, and H₂, and 6) one or morecarbohydrates. Exemplary carbohydrates include, but are not limited to,glucose, sucrose, xylose, arabinose, and glycerol.

The enzymes used for the conversion of CO, CO₂, and/or H₂ to acetyl-CoAare shown in FIG. 5. To engineer a production host for the utilizationof CO, CO₂, and/or H₂, one or more exogenous DNA sequence(s) encodingthese enzymes can be expressed in the microorganism. Referring now toFIG. 5, described below are enzymes that can be incorporated to utilizeCO, CO₂, and/or H₂.

Formate dehydrogenase is a two subunit selenocysteine-containing proteinthat catalyzes the incorporation of CO₂ into formate in Moorellathermoacetica (Andreesen and Ljungdahl, J. Bacteriol. 116.2:867-873(1973); Li et al., J. Bacteriol. 92.2:405-412 (1966); Yamamoto et al. J.Biol. Chem. 258.3:1826-1832 (1983)). The loci, Moth_(—)2312 andMoth_(—)2313 are actually one gene that is responsible for encoding thealpha subunit of formate dehydrogenase while the beta subunit is encodedby Moth_(—)2314 (Pierce et al. Environ. Microbiol. 10:2550-2573 (2008)).Another set of genes encoding formate dehydrogenase activity with apropensity for CO₂ reduction is encoded by Sfum_(—)2703 throughSfum_(—)2706 in Syntrophobacter fumaroxidans (Reda et al., Proc. Natl.Acad. Sci. U.S.A. 105.31:10654-10658 (2008); de Bok et al., Eur. J.Biochem. 270. 11: 2476-2485 (2003)). Similar to their M. thermoaceticacounterparts, Sfum_(—)2705 and Sfum_(—)2706 are actually one gene. Asimilar set of genes presumed to carry out the same function are encodedby CHY_(—)0731, CHY_(—)0732, and CHY_(—)0733 in C. hydrogenoformans (Wuet al., PLoS Genet. 1.5:e65 (2005)). Relevant gene and proteininformation can be obtained from the information shown below:

Protein GenBank ID GI number Organism Moth_2312 YP_431142 148283121Moorella thermoacetica Moth_2313 YP_431143 Moorella thermoaceticaMoth_2314 YP_431144 83591135 Moorella thermoacetica Sfum_2703YP_846816.1 116750129 Syntrophobacter fumaroxidans Sfum_2704 YP_846817.1116750130 Syntrophobacter fumaroxidans Sfum_2705 YP_846818.1 116750131Syntrophobacter fumaroxidans Sfum_2706 YP_846819.1 116750132Syntrophobacter fumaroxidans CHY_0731 YP_359585.1 78044572Carboxydothermus hydrogenoformans CHY_0732 YP_359586.1 78044500Carboxydothermus hydrogenoformans CHY_0733 YP_359587.1 78044647Carboxydothermus hydrogenoformans

Formyltetrahydrofolate synthetase ligates formate to tetrahydrofolate atthe expense of one ATP. This reaction is catalyzed by the gene productof Moth_(—)0109 in M. thermoacetica (O'brien et al., Experientia Suppl.26:249-262 (1976); Lovell et al., Arch. Microbiol. 149.4:280-285 (1988);Lovell et al., Biochemistry 29.24:5687-5694 (1990)), FHS in Clostridiumacidurici (Whitehead and Rabinowitz, J. Bacteriol. 167.1:205-209 (1986);Whitehead and Rabinowitz, J. Bacteriol. 170.7:3255-3261 (1988)), andCHY_(—)2385 in C. hydrogenoformans (Wu et al., PLoS Genet. 1.5:e65(2005)). Relevant gene and protein information can be obtained from theinformation shown below:

Protein GenBank ID GI number Organism Moth_0109 YP_428991.1 83588982Moorella thermoacetica CHY_2385 YP_361182.1 78045024 Carboxydothermushydrogenoformans FHS P13419.1 120562 Clostridium acidurici

In M. thermoacetica, E. coli, and C. hydrogenoformans,methenyltetrahydrofolate cyclohydrolase and methylenetetrahydrofolatedehydrogenase are carried out by the bi-functional gene products ofMoth_(—)1516, folD, and CHY_(—)1878, respectively (Pierce et al.Environ. Microbiol. 10:2550-2573 (2008); (Wu et al., PLoS Genet. 1.5:e65(2005); D'Ari and Rabinowitz, J. Biol. Chem. 266.35:23953-23958 (1991)).Relevant gene and protein information can be obtained from theinformation shown below:

Protein GenBank ID GI number Organism Moth_1516 YP_430368.1 83590359Moorella thermoacetica folD NP_415062.1 16128513 Escherichia coliCHY_1878 YP_360698.1 78044829 Carboxydothermus hydrogenoformans

In M. thermoacetica, E. coli, and C. hydrogenoformans,methenyltetrahydrofolate cyclohydrolase and methylenetetrahydrofolatedehydrogenase are carried out by the bi-functional gene products ofMoth_(—)1516, folD, and CHY_(—)1878, respectively (Pierce et al.Environ. Microbiol. 10:2550-2573 (2008); (Wu et al., PLoS Genet. 1.5:e65(2005); D'Ari and Rabinowitz, J. Biol. Chem. 266.35:23953-23958 (1991)).Relevant gene and protein information can be obtained from theinformation shown below:

Protein GenBank ID GI number Organism Moth_1516 YP_430368.1 83590359Moorella thermoacetica folD NP_415062.1 16128513 Escherichia coliCHY_1878 YP_360698.1 78044829 Carboxydothermus hydrogenoformans

The final step of the methyl branch of the Wood-Ljungdahl pathway iscatalyzed by methylenetetrahydrofolate reductase. In M. thermoacetica,this enzyme is oxygen-sensitive and contains an iron-sulfur cluster(Clark and Ljungdahl, J. Biol. Chem. 259.17:10845-10849 (1984)). Thisenzyme is encoded by metF in E. coli (Sheppard et al., J. Bacteriol.181.3:718-725 (1999)) and CHY_(—)1233 in C. hydrogenoformans (Wu et al.,PLoS Genet. 1.5:e65 (2005)). The M. thermoacetica genes, and its C.hydrogenoformans counterpart, are located near the CODH/ACS genecluster, separated by putative hydrogenase and heterodisulfide reductasegenes.

Protein GenBank ID GI number Organism Moth_1191 YP_430048.1 83590039Moorella thermoacetica metF NP_418376.1 16131779 Escherichia coliCHY_1233 YP_360071.1 78044792 Carboxydothermus hydrogenoformans

While E. coli naturally possesses the capability for some of therequired transformations (i.e., methenyltetrahydrofolate cyclohydrolase,methylenetetrahydrofolate dehydrogenase, methylenetetrahydrofolatereductase), the methyl branch enzymes from acetogens can havesignificantly higher (50-100×) specific activities than those fromnon-acetogens (Morton et al., Genetics and molecular biology ofanaerobic bacteria, Ed. M. Sebald, New York: Springer Verlag (1992)pages 389-406). Formate dehydrogenase may be specialized for anaerobicconditions (Ljungdahl and Andreesen, FEBS Lett. 54.2:279-282 (1975))(1975). Therefore, various non-native versions of each of these can beexpressed in the strain of E. coli capable of methanol and CO₂, CO,and/or H₂ utilization. Specifically, these genes can be cloned andcombined into an expression vector designed to express them as a set.Initially, a high or medium copy number vector can be chosen (usingColE1 or P15A replicons). An exemplary promoter is a stronglyconstitutive promoter such as lambda pL or an IPTG-inducible version ofthis, pL-lacO (Lutz and Bujard, Nucleic Acids Res. 25.6:1203-1210(1997)). To make an artificial operon, one 5′ terminal promoter isplaced upstream of the set of genes and each gene receives a consensusrbs element. The order of genes is based on the natural order wheneverpossible. Ultimately, the genes are integrated into the E. colichromosome. Enzyme assays are performed as described in (Ljungdahl andAndreesen, Methods Enzymol. 53:360-372 (1978); Yamamoto et al. J. Biol.Chem. 258.3:1826-1832 (1983); Lovell et al., Arch. Microbiol.149.4:280-285 (1988); de Mata and Rabinowitz, J. Biol. Chem.255.6:2569-2577 (1980); D'Ari and Rabinowitz, J. Biol. Chem.266.35:23953-23958 (1991); Clark and Ljungdahl, 259.17:10845-10849(1984); Clark and Ljungdahl, Methods Enzymol. 122:392-399 (1986)).

After strains of E. coli expressing both the carbonyl and methylbranches of the Wood-Ljungdahl pathway are constructed, they are assayedfor the ability to utilize CO, CO₂, and/or H₂, for incorporation intoacetyl-CoA, cell mass, 1,3-butanediol. Initial conditions employstrictly anaerobically grown cells provided with exogenous glucose.Metabolizing glucose or other carbohydrates to acetyl-CoA provides onepotential source of CO₂ that can be fixed via the Wood-Ljungdahlpathway. Alternatively, or in addition to glucose, nitrate can be addedto the fermentation broth to serve as an electron acceptor and initiatorof growth. Anaerobic growth of E. coli on fatty acids, which areultimately metabolized to acetyl-CoA, has been demonstrated in thepresence of nitrate (Campbell et al., Mol. Microbiol. 47.3:793-805(2003)). Oxygen can also be provided as long as its intracellular levelsare maintained below any inhibition threshold of the engineered enzymes.‘Synthetic syngas’ of a composition suitable for these experiments canalso be employed. ¹³C-labeled CO and/or CO₂ are provided to the cellsand analytical mass spectrometry is employed to measure incorporation ofthe labeled carbon into acetate, 1,3-butanediol, and cell mass (e.g.,proteinogenic amino acids).

Process considerations for a syngas fermentation include high biomassconcentration and good gas-liquid mass transfer (Bredwell et al.,Biotechnol. Prog. 15.5:834-844 (1999)). The solubility of CO in water issomewhat less than that of oxygen. Continuously gas-spargedfermentations can be performed in controlled fermenters with constantoff-gas analysis by mass spectrometry and periodic liquid sampling andanalysis by GC and HPLC. The liquid phase can function in batch mode.Fermentation products such as alcohols, organic acids, and residualglucose along with residual methanol are quantified by HPLC (Shimadzu,Columbia Md.), for example, using an Aminex® series of HPLC columns (forexample, HPX-87 series) (BioRad, Hercules Calif.), using a refractiveindex detector for glucose and alcohols, and a UV detector for organicacids. The growth rate is determined by measuring optical density usinga spectrophotometer (600 nm). All piping in these systems is glass ormetal to maintain anaerobic conditions. The gas sparging can beperformed with glass frits to decrease bubble size and improve masstransfer. Various sparging rates are tested, ranging from about 0.1 to 1vvm (vapor volumes per minute). To obtain accurate measurements of gasuptake rates, periodic challenges are performed in which the gas flow istemporarily stopped, and the gas phase composition is monitored as afunction of time.

In order to achieve the overall target productivity, methods of cellretention or recycle can be employed. One method to increase themicrobial concentration is to recycle cells via a tangential flowmembrane from a sidestream. Repeated batch culture can also be used, aspreviously described for production of acetate by Moorella (Sakai etal., J. Biosci. Bioeng. 99.3:252-258 (2005)). Various other methods canalso be used (Bredwell et al., Biotechnol. Prog. 15.5:834-844 (1999);Datar et al., Biotechnol. Bioeng. 86.5:587-594 (2004)). Additionaloptimization can be tested such as overpressure at 1.5 atm to improvemass transfer (Najafpour and Younesi, Enzyme and Microbial Technology38:223-228 (2006)).

Once satisfactory performance is achieved using pure H₂/CO as the feed,synthetic gas mixtures can be generated containing inhibitors likely tobe present in commercial syngas. For example, a typical impurity profileis 4.5% CH₄, 0.1% C₂H₂, 0.35% C₂H₆, 1.4% C₂H₄, and 150 ppm nitric oxide(Datar et al., Biotechnol. Bioeng. 86.5:587-594 (2004)). Tars,represented by compounds such as benzene, toluene, ethylbenzene,p-xylene, o-xylene, and naphthalene, are added at ppm levels to test forany effect on production. For example, it has been shown that 40 ppm NOis inhibitory to C. carboxidivorans (Ahmed and Lewis, Biotechnol.Bioeng. 97.5:1080-1086 (2007)). Cultures can be tested in shake-flaskcultures before moving to a fermentor. Also, different levels of thesepotential inhibitory compounds are tested to quantify the effect theyhave on cell growth. This knowledge is used to develop specificationsfor syngas purity, which is utilized for scale up studies andproduction. If any particular component is found to be difficult todecrease or remove from syngas used for scale up, an adaptive evolutionprocedure is utilized to adapt cells to tolerate one or more impurities.

The invention is described herein with general reference to themetabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing, or a protein associated with, thereferenced metabolic reaction, reactant or product. Unless otherwiseexpressly stated herein, those skilled in the art will understand thatreference to a reaction also constitutes reference to the reactants andproducts of the reaction. Similarly, unless otherwise expressly statedherein, reference to a reactant or product also references the reaction,and reference to any of these metabolic constituents also references thegene or genes encoding the enzymes that catalyze or proteins involved inthe referenced reaction, reactant or product. Likewise, given the wellknown fields of metabolic biochemistry, enzymology and genomics,reference herein to a gene or encoding nucleic acid also constitutes areference to the corresponding encoded enzyme and the reaction itcatalyzes or a protein associated with the reaction as well as thereactants and products of the reaction.

The non-naturally occurring microbial organisms of the invention can beproduced by introducing expressible nucleic acids encoding one or moreof the enzymes or proteins participating in one or more 1,3-butanediolbiosynthetic pathways. Depending on the host microbial organism chosenfor biosynthesis, nucleic acids for some or all of a particular1,3-butanediol biosynthetic pathway can be expressed. For example, if achosen host is deficient in one or more enzymes or proteins for adesired biosynthetic pathway, then expressible nucleic acids for thedeficient enzyme(s) or protein(s) are introduced into the host forsubsequent exogenous expression. Alternatively, if the chosen hostexhibits endogenous expression of some pathway genes, but is deficientin others, then an encoding nucleic acid is needed for the deficientenzyme(s) or protein(s) to achieve 1,3-butanediol biosynthesis. Thus, anon-naturally occurring microbial organism of the invention can beproduced by introducing exogenous enzyme or protein activities to obtaina desired biosynthetic pathway or a desired biosynthetic pathway can beobtained by introducing one or more exogenous enzyme or proteinactivities that, together with one or more endogenous enzymes orproteins, produces a desired product such as 1,3-butanediol.

Host microbial organisms can be selected from, and the non-naturallyoccurring microbial organisms generated in, for example, bacteria,yeast, fungus, algae, cyanobacteria, or any of a variety of othermicroorganisms applicable to fermentation processes. Exemplary bacteriainclude species selected from Escherichia coli, Klebsiella oxytoca,Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes,Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis,Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis,Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor,Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonasputida. Exemplary yeasts or fungi include species selected fromSaccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyceslactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger,Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae, and the like.Exemplary cyanobacteria include Acaryochloris marina MBIC11017, Anabaenasp. PCC 7120, Anabaena variabilis ATCC 29413, Agmenellum quadruplicatum,Chlorobium tepidum TLS, Cyanothece sp. ATCC 51142, Gloeobacter violaceusPCC 7421, Microcystis aeruginosa NIES-843, Nostoc punctiforme ATCC29133, Prochlorococcus marinus MED4, Prochlorococcus marinus MIT9313,Prochlorococcus marinus SS120, Prochlorococcus marinus str. AS9601,Prochlorococcus marinus str. MIT 9211, Prochlorococcus marinus str. MIT9215, Prochlorococcus marinus str. MIT 9301, Prochlorococcus marinusstr. MIT 9303, Prochlorococcus marinus str. MIT 9312, Prochlorococcusmarinus str. MIT 9515, Prochlorococcus marinus str. NATL1A,Prochlorococcus marinus str. NATL2A, Rhodopseudomonas palustris CGA009,Synechococcus elongatus PCC 6301, Synechococcus elongatus PCC 7942,Synechococcus sp. CC9311, Synechococcus sp. CC9605, Synechococcus sp.CC9902, Synechococcus sp. JA-2-3B\'a(2-13), Synechococcus sp. JA-3-3Ab,Synechococcus sp. PCC 7002, Synechococcus sp. RCC307, Synechococcus sp.WH 7803, Synechococcus sp. WH8102, Synechocystis sp. PCC 6803,Thermosynechococcus elongatus BP-1, Trichodesmium erythraeum IMS101.Exemplary algae include Botryococcus braunii, Chlamydomonas reinhardii,Chlorella sp., Crypthecodinium cohnii, Cylindrotheca sp., Dunaliellaprimolecta, Isochrysis sp., Monallanthus salina, Nannochloris sp.,Nannochloropsis sp., Neochloris oleoabundans, Nitzschia sp.,Phaeodactylum tricornutum, Schizochytrium sp., Tetraselmis sueica. E.coli is a particularly useful host organisms since it is a wellcharacterized microbial organism suitable for genetic engineering. Otherparticularly useful host organisms include yeast such as Saccharomycescerevisiae. It is understood that any suitable microbial host organismcan be used to introduce metabolic and/or genetic modifications toproduce a desired product.

Depending on the 1,3-butanediol biosynthetic pathway constituents of aselected host microbial organism, the non-naturally occurring microbialorganisms of the invention will include at least one exogenouslyexpressed 1,3-BDO pathway-encoding nucleic acid and up to all encodingnucleic acids for one or more 1,3-butanediol biosynthetic pathways. Forexample, 1,3-butanediol biosynthesis can be established in a hostdeficient in a pathway enzyme or protein through exogenous expression ofthe corresponding encoding nucleic acid. In a host deficient in allenzymes or proteins of a 1,3-butanediol pathway, exogenous expression ofall enzyme or proteins in the pathway can be included, although it isunderstood that all enzymes or proteins of a pathway can be expressedeven if the host contains at least one of the pathway enzymes orproteins. For example, exogenous expression of all enzymes or proteinsin a pathway for production of 1,3-butanediol can be included, suchas 1) Methanol methyltransferase (MtaB), 2) Corrinoid protein (MtaC), 3)Methyltetrahydrofolate:corrinoid protein methyltransferase (MtaA), 4)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 5)Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein(AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB &AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11)Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA reductase(CoA-dependent, alcohol forming), 13) 3-oxobutyraldehyde reductase(aldehyde reducing), 14) 4-hydroxy,2-butanone reductase, 15)Acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming), 16)3-oxobutyraldehyde reductase (ketone reducing), 17)3-hydroxybutyraldehyde reductase, 18) Acetoacetyl-CoA reductase (ketonereducing), 19) 3-hydroxybutyryl-CoA reductase (aldehyde forming), 20)3-hydroxybutyryl-CoA reductase (alcohol forming), 21) acetoacetyl-CoAtransferase, hydrolase, or synthetase, 22) acetoacetate reductase, 23)3-hydroxybutyryl-CoA transferase, hydrolase, or synthetase, 24)3-hydroxybutyrate reductase, and 25) 3-hydroxybutyrate dehydrogenase, asshown in FIG. 4.

Alternatively, exogenous expression of all enzymes or proteins in apathway for production of 1,3-butanediol, as shown in FIG. 5, can beincluded, such as 1) Formate dehydrogenase, 2) Formyltetrahydrofolatesynthetase, 3) Methenyltetrahydrofolate cyclohydrolase, 4)Methylenetetrahydrofolate dehydrogenase, 5) Methylenetetrahydrofolatereductase, 6) Methyltetrahydrofolate:corrinoid protein methyltransferase(AcsE), 7) Corrinoid iron-sulfur protein (AcsD), 8) Nickel-proteinassembly protein (AcsF & CooC), 9) Ferredoxin (Orf7), 10) Acetyl-CoAsynthase (AcsB & AcsC), 11) Carbon monoxide dehydrogenase (AcsA), 12)Hydrogenase (Hyd), 13) Acetoacetyl-CoA thiolase (AtoB), 14)Acetoacetyl-CoA reductase (CoA-dependent, alcohol forming), 15)3-oxobutyraldehyde reductase (aldehyde reducing), 16)4-hydroxy,2-butanone reductase, 17) Acetoacetyl-CoA reductase(CoA-dependent, aldehyde forming), 18) 3-oxobutyraldehyde reductase(ketone reducing), 19) 3-hydroxybutyraldehyde reductase, 20)Acetoacetyl-CoA reductase (ketone reducing), 21) 3-hydroxybutyryl-CoAreductase (aldehyde forming), 22) 3-hydroxybutyryl-CoA reductase(alcohol forming), 23) acetoacetyl-CoA transferase, hydrolase, orsynthetase, 24) acetoacetate reductase, 25) 3-hydroxybutyryl-CoAtransferase, hydrolase, or synthetase, 26) 3-hydroxybutyrate reductase,and 27) 3-hydroxybutyrate dehydrogenase.

Given the teachings and guidance provided herein, those skilled in theart will understand that the number of encoding nucleic acids tointroduce in an expressible form will, at least, parallel the1,3-butanediol pathway deficiencies of the selected host microbialorganism. Therefore, a non-naturally occurring microbial organism of theinvention can have one, two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeenthat is, up to all nucleic acids encoding the enzymes or proteinsconstituting a 1,3-butanediol biosynthetic pathway disclosed herein andshown in FIGS. 4 and 5. In some embodiments, the non-naturally occurringmicrobial organisms also can include other genetic modifications thatfacilitate or optimize 1,3-butanediol biosynthesis or that confer otheruseful functions onto the host microbial organism. One such otherfunctionality can include, for example, augmentation of the synthesis ofone or more of the 1,3-butanediol pathway precursors such as acetyl-CoA,acetoacetyl-CoA, acetoacetate, 3-hydroxybutyryl-CoA, 3-hydroxybutyrate,4-hydroxy-2-butanone, 3-oxobutryaldehyde, or 3-hydroxybutryaldehyde.

Generally, a host microbial organism is selected such that it producesthe precursor of a 1,3-butanediol pathway, either as a naturallyproduced molecule or as an engineered product that either provides denovo production of a desired precursor or increased production of aprecursor naturally produced by the host microbial organism. Forexample, acetyl-CoA is produced naturally in a host organism such as E.coli. A host organism can be engineered to increase production of aprecursor, as disclosed herein. In addition, a microbial organism thathas been engineered to produce a desired precursor can be used as a hostorganism and further engineered to express enzymes or proteins of a1,3-butanediol pathway.

In some embodiments, a non-naturally occurring microbial organism of theinvention is generated from a host that contains the enzymaticcapability to synthesize 1,3-butanediol. In this specific embodiment itcan be useful to increase the synthesis or accumulation of a1,3-butanediol pathway product to, for example, drive 1,3-butanediolpathway reactions toward 1,3-butanediol production. Increased synthesisor accumulation can be accomplished by, for example, overexpression ofnucleic acids encoding one or more of the above-described 1,3-butanediolpathway enzymes or proteins. Over expression the enzyme or enzymesand/or protein or proteins of the 1,3-butanediol pathway can occur, forexample, through exogenous expression of the endogenous gene or genes,or through exogenous expression of the heterologous gene or genes.Therefore, naturally occurring organisms can be readily generated to benon-naturally occurring microbial organisms of the invention, forexample, producing 1,3-butanediol, through overexpression of one, two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen that is, up to allnucleic acids encoding the enzymes or proteins constituting a1,3-butanediol biosynthetic pathway disclosed herein and shown in FIGS.4 and 5. In addition, a non-naturally occurring organism can begenerated by mutagenesis of an endogenous gene that results in anincrease in activity of an enzyme in the 1,3-butanediol biosyntheticpathway.

In particularly useful embodiments, exogenous expression of the encodingnucleic acids is employed. Exogenous expression confers the ability tocustom tailor the expression and/or regulatory elements to the host andapplication to achieve a desired expression level that is controlled bythe user. However, endogenous expression also can be utilized in otherembodiments such as by removing a negative regulatory effector orinduction of the gene's promoter when linked to an inducible promoter orother regulatory element. Thus, an endogenous gene having a naturallyoccurring inducible promoter can be up-regulated by providing theappropriate inducing agent, or the regulatory region of an endogenousgene can be engineered to incorporate an inducible regulatory element,thereby allowing the regulation of increased expression of an endogenousgene at a desired time. Similarly, an inducible promoter can be includedas a regulatory element for an exogenous gene introduced into anon-naturally occurring microbial organism.

It is understood that, in methods of the invention, any of the one ormore exogenous nucleic acids can be introduced into a microbial organismto produce a non-naturally occurring microbial organism of theinvention. The nucleic acids can be introduced so as to confer, forexample, a 1,3-butanediol biosynthetic pathway onto the microbialorganism. Alternatively, encoding nucleic acids can be introduced toproduce an intermediate microbial organism having the biosyntheticcapability to catalyze some of the required reactions to confer1,3-butanediol biosynthetic capability. For example, a non-naturallyoccurring microbial organism having a 1,3-butanediol biosyntheticpathway can comprise at least two exogenous nucleic acids encodingdesired enzymes or proteins. Thus, it is understood that any combinationof two or more enzymes or proteins of a biosynthetic pathway can beincluded in a non-naturally occurring microbial organism of theinvention. Similarly, it is understood that any combination of three ormore enzymes or proteins of a biosynthetic pathway can be included in anon-naturally occurring microbial organism of the invention. Similarly,any combination of four, or more enzymes or proteins of a biosyntheticpathway as disclosed herein can be included in a non-naturally occurringmicrobial organism of the invention, as desired, so long as thecombination of enzymes and/or proteins of the desired biosyntheticpathway results in production of the corresponding desired product.Likewise, any combination of five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, fifteen, sixteen, enzymes and/or proteins ofa biosynthetic pathway as disclosed herein can be included in anon-naturally occurring microbial organism of the invention, as desired,so long as the combination of enzymes and/or proteins of the desiredbiosynthetic pathway results in production of the corresponding desiredproduct.

Exemplary combinations of 17 exogenous enzymes or proteins of abiosynthetic pathway, as disclosed herein, included in a non-naturallyoccurring microbial organism of the invention include:

A: 1) Formate dehydrogenase, 2) Formyltetrahydrofolate synthetase, 3)Methenyltetrahydrofolate cyclohydrolase, 4) Methylenetetrahydrofolatedehydrogenase, 5) Methylenetetrahydrofolate reductase, 6)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 7)Corrinoid iron-sulfur protein (AcsD), 8) Nickel-protein assembly protein(AcsF & CooC), 9) Ferredoxin (Orf7), 10) Acetyl-CoA synthase (AcsB &AcsC), 11) Carbon monoxide dehydrogenase (AcsA), 12) Hydrogenase (Hyd),13) Acetoacetyl-CoA thiolase (AtoB), 14) Acetoacetyl-CoA transferase,hydrolase, or synthetase, 15) Acetoacetate reductase, 16)3-oxobutyraldehyde reductase (ketone reducing), 17)3-hydroxybutyraldehyde reductase;

B: 1) Formate dehydrogenase, 2) Formyltetrahydrofolate synthetase, 3)Methenyltetrahydrofolate cyclohydrolase, 4) Methylenetetrahydrofolatedehydrogenase, 5) Methylenetetrahydrofolate reductase, 6)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 7)Corrinoid iron-sulfur protein (AcsD), 8) Nickel-protein assembly protein(AcsF & CooC), 9) Ferredoxin (Orf7), 10) Acetyl-CoA synthase (AcsB &AcsC), 11) Carbon monoxide dehydrogenase (AcsA), 12) Hydrogenase (Hyd),13) Acetoacetyl-CoA thiolase (AtoB), 14) Acetoacetyl-CoA transferase,hydrolase, or synthetase, 15) Acetoacetate reductase, 16)3-oxobutyraldehyde reductase (aldehyde reducing), 17)4-hydroxy,2-butanone reductase;

C: 1) Formate dehydrogenase, 2) Formyltetrahydrofolate synthetase, 3)Methenyltetrahydrofolate cyclohydrolase, 4) Methylenetetrahydrofolatedehydrogenase, 5) Methylenetetrahydrofolate reductase, 6)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 7)Corrinoid iron-sulfur protein (AcsD), 8) Nickel-protein assembly protein(AcsF & CooC), 9) Ferredoxin (Orf7), 10) Acetyl-CoA synthase (AcsB &AcsC), 11) Carbon monoxide dehydrogenase (AcsA), 12) Hydrogenase (Hyd),13) Acetoacetyl-CoA thiolase (AtoB), 14) Acetoacetyl-CoA reductase(ketone reducing), 15) 3-hydroxybutyryl-CoA transferase, hydrolase, orsynthetase, 16) 3-hydroxybutyrate reductase, 17) 3-hydroxybutyraldehydereductase;

D: 1) Formate dehydrogenase, 2) Formyltetrahydrofolate synthetase, 3)Methenyltetrahydrofolate cyclohydrolase, 4) Methylenetetrahydrofolatedehydrogenase, 5) Methylenetetrahydrofolate reductase, 6)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 7)Corrinoid iron-sulfur protein (AcsD), 8) Nickel-protein assembly protein(AcsF & CooC), 9) Ferredoxin (Orf7), 10) Acetyl-CoA synthase (AcsB &AcsC), 11) Carbon monoxide dehydrogenase (AcsA), 12) Hydrogenase (Hyd),13) Acetoacetyl-CoA thiolase (AtoB), 14) Acetoacetyl-CoA transferase,hydrolase, or synthetase, 15) 3-hydroxybutyrate dehydrogenase, 16)3-hydroxybutyrate reductase, 17) 3-hydroxybutyraldehyde reductase.

Exemplary combinations of 16 exogenous enzymes or proteins of abiosynthetic pathway, as disclosed herein, included in a non-naturallyoccurring microbial organism of the invention include any combination of16 of the 17 enzymes disclosed above in A-D or:

E: 1) Formate dehydrogenase, 2) Formyltetrahydrofolate synthetase, 3)Methenyltetrahydrofolate cyclohydrolase, 4) Methylenetetrahydrofolatedehydrogenase, 5) Methylenetetrahydrofolate reductase, 6)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 7)Corrinoid iron-sulfur protein (AcsD), 8) Nickel-protein assembly protein(AcsF & CooC), 9) Ferredoxin (Orf7), 10) Acetyl-CoA synthase (AcsB &AcsC), 11) Carbon monoxide dehydrogenase (AcsA), 12) Hydrogenase (Hyd),13) Acetoacetyl-CoA thiolase (AtoB), 14) Acetoacetyl-CoA reductase(CoA-dependent, aldehyde forming), 15) 3-oxobutyraldehyde reductase(aldehyde reducing), and 16) 4-hydroxy,2-butanone reductase.

F: 1) Formate dehydrogenase, 2) Formyltetrahydrofolate synthetase, 3)Methenyltetrahydrofolate cyclohydrolase, 4) Methylenetetrahydrofolatedehydrogenase, 5) Methylenetetrahydrofolate reductase, 6)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 7)Corrinoid iron-sulfur protein (AcsD), 8) Nickel-protein assembly protein(AcsF & CooC), 9) Ferredoxin (Orf7), 10) Acetyl-CoA synthase (AcsB &AcsC), 11) Carbon monoxide dehydrogenase (AcsA), 12) Hydrogenase (Hyd),13) Acetoacetyl-CoA thiolase (AtoB), 14) Acetoacetyl-CoA reductase(CoA-dependent, aldehyde forming), 15) 3-oxobutyraldehyde reductase(ketone reducing), and 16) 3-hydroxybutyraldehyde reductase.

G: 1) Formate dehydrogenase, 2) Formyltetrahydrofolate synthetase, 3)Methenyltetrahydrofolate cyclohydrolase, 4) Methylenetetrahydrofolatedehydrogenase, 5) Methylenetetrahydrofolate reductase, 6)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 7)Corrinoid iron-sulfur protein (AcsD), 8) Nickel-protein assembly protein(AcsF & CooC), 9) Ferredoxin (Orf7), 10) Acetyl-CoA synthase (AcsB &AcsC), 11) Carbon monoxide dehydrogenase (AcsA), 12) Hydrogenase (Hyd),13) Acetoacetyl-CoA thiolase (AtoB), 14) Acetoacetyl-CoA reductase(ketone reducing), 15) 3-hydroxybutyryl-CoA reductase (aldehydeforming), and 16) 3-hydroxybutyraldehyde reductase.

Exemplary combinations of 15 exogenous enzymes or proteins of abiosynthetic pathway, as disclosed herein, included in a non-naturallyoccurring microbial organism of the invention any combination of 15 ofthe enzymes disclosed above in A-G or:

H: 1) Formate dehydrogenase, 2) Formyltetrahydrofolate synthetase, 3)Methenyltetrahydrofolate cyclohydrolase, 4) Methylenetetrahydrofolatedehydrogenase, 5) Methylenetetrahydrofolate reductase, 6)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 7)Corrinoid iron-sulfur protein (AcsD), 8) Nickel-protein assembly protein(AcsF & CooC), 9) Ferredoxin (Orf7), 10) Acetyl-CoA synthase (AcsB &AcsC), 11) Carbon monoxide dehydrogenase (AcsA), 12) Hydrogenase (Hyd),13) Acetoacetyl-CoA thiolase (AtoB), 14) Acetoacetyl-CoA reductase(CoA-dependent, alcohol forming), and 15) 4-hydroxy,2-butanonereductase.

I: 1) Formate dehydrogenase, 2) Formyltetrahydrofolate synthetase, 3)Methenyltetrahydrofolate cyclohydrolase, 4) Methylenetetrahydrofolatedehydrogenase, 5) Methylenetetrahydrofolate reductase, 6)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 7)Corrinoid iron-sulfur protein (AcsD), 8) Nickel-protein assembly protein(AcsF & CooC), 9) Ferredoxin (Orf7), 10) Acetyl-CoA synthase (AcsB &AcsC), 11) Carbon monoxide dehydrogenase (AcsA), 12) Hydrogenase (Hyd),13) Acetoacetyl-CoA thiolase (AtoB), 14) Acetoacetyl-CoA reductase(ketone reducing), and 15) 3-hydroxybutyryl-CoA reductase (alcoholforming).

J: 1) Methanol methyltransferase (MtaB), 2) Corrinoid protein (MtaC), 3)Methyltetrahydrofolate:corrinoid protein methyltransferase (MtaA), 4)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 5)Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein(AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB &AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11)Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA transferase,hydrolase, or synthetase, 13) Acetoacetate reductase, 14)3-oxobutyraldehyde reductase (ketone reducing), 15)3-hydroxybutyraldehyde reductase;

K: 1) Methanol methyltransferase (MtaB), 2) Corrinoid protein (MtaC), 3)Methyltetrahydrofolate:corrinoid protein methyltransferase (MtaA), 4)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 5)Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein(AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB &AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11)Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA transferase,hydrolase, or synthetase, 13) Acetoacetate reductase, 14)3-oxobutyraldehyde reductase (aldehyde reducing), 15)4-hydroxy,2-butanone reductase;

L: 1) Methanol methyltransferase (MtaB), 2) Corrinoid protein (MtaC), 3)Methyltetrahydrofolate:corrinoid protein methyltransferase (MtaA), 4)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 5)Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein(AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB &AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11)Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA reductase (ketonereducing), 13) 3-hydroxybutyryl-CoA transferase, hydrolase, orsynthetase, 14) 3-hydroxybutyrate reductase, 15) 3-hydroxybutyraldehydereductase;

M: 1) Methanol methyltransferase (MtaB), 2) Corrinoid protein (MtaC), 3)Methyltetrahydrofolate:corrinoid protein methyltransferase (MtaA), 4)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 5)Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein(AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB &AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11)Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA transferase,hydrolase, or synthetase, 13) 3-hydroxybutyrate dehydrogenase, 14)3-hydroxybutyrate reductase, 15) 3-hydroxybutyraldehyde reductase.

Exemplary combinations of 14 exogenous enzymes or proteins of abiosynthetic pathway, as disclosed herein, included in a non-naturallyoccurring microbial organism of the invention include any combination of14 of the enzymes disclosed above in A-M or:

N: 1) Methanol methyltransferase (MtaB), 2) Corrinoid protein (MtaC), 3)Methyltetrahydrofolate:corrinoid protein methyltransferase (MtaA), 4)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 5)Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein(AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB &AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11)Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA reductase(CoA-dependent, aldehyde forming)13) 3-oxobutyraldehyde reductase(aldehyde reducing), and 14) 4-hydroxy,2-butanone reductase.

O: 1) Methanol methyltransferase (MtaB), 2) Corrinoid protein (MtaC), 3)Methyltetrahydrofolate:corrinoid protein methyltransferase (MtaA), 4)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 5)Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein(AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB &AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11)Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA reductase(CoA-dependent, aldehyde forming), 13) 3-oxobutyraldehyde reductase(ketone reducing), and 14) 3-hydroxybutyraldehyde reductase.

P: 1) Methanol methyltransferase (MtaB), 2) Corrinoid protein (MtaC), 3)Methyltetrahydrofolate:corrinoid protein methyltransferase (MtaA), 4)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 5)Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein(AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB &AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11)Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA reductase (ketonereducing), 13) 3-hydroxybutyryl-CoA reductase (aldehyde forming), and14) 3-hydroxybutyraldehyde reductase

Exemplary combinations of 13 exogenous enzymes or proteins of abiosynthetic pathway, as disclosed herein, included in a non-naturallyoccurring microbial organism of the invention include any combination of13 of the enzymes disclosed above in A-P or:

Q: 1) Methanol methyltransferase (MtaB), 2) Corrinoid protein (MtaC), 3)Methyltetrahydrofolate:corrinoid protein methyltransferase (MtaA), 4)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 5)Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein(AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB &AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11)Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA reductase (ketonereducing), and 13) 3-hydroxybutyryl-CoA reductase (alcohol forming),

R: 1) Methanol methyltransferase (MtaB), 2) Corrinoid protein (MtaC), 3)Methyltetrahydrofolate:corrinoid protein methyltransferase (MtaA), 4)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 5)Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein(AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB &AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11)Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA reductase(CoA-dependent, alcohol forming), and 13) 4-hydroxy,2-butanonereductase.

Exemplary combinations of 12 exogenous enzymes or proteins of abiosynthetic pathway, as disclosed herein, included in a non-naturallyoccurring microbial organism of the invention include any combination of12 of the enzymes disclosed above in A-R. Exemplary combinations of 11exogenous enzymes or proteins of a biosynthetic pathway, as disclosedherein, included in a non-naturally occurring microbial organism of theinvention include any combination of 10 of the enzymes disclosed abovein A-R. Exemplary combinations of 9 exogenous enzymes or proteins of abiosynthetic pathway, as disclosed herein, included in a non-naturallyoccurring microbial organism of the invention include any combination of8 of the enzymes disclosed above in A-R. Exemplary combinations of 7exogenous enzymes or proteins of a biosynthetic pathway, as disclosedherein, included in a non-naturally occurring microbial organism of theinvention include any combination of 6 of the enzymes disclosed above inA-R, and so on down to any combination of 2 of the enzymes disclosedabove in A-R.

In addition to the biosynthesis of 1,3-butanediol as described herein,the non-naturally occurring microbial organisms and methods of theinvention also can be utilized in various combinations with each otherand with other microbial organisms and methods well known in the art toachieve product biosynthesis by other routes. For example, onealternative to produce 1,3-butanediol other than use of the1,3-butanediol producers is through addition of another microbialorganism capable of converting a 1,3-butanediol pathway intermediate to1,3-butanediol. One such procedure includes, for example, thefermentation of a microbial organism that produces a 1,3-butanediolpathway intermediate. The 1,3-butanediol pathway intermediate can thenbe used as a substrate for a second microbial organism that converts the1,3-butanediol pathway intermediate to 1,3-butanediol. The1,3-butanediol pathway intermediate can be added directly to anotherculture of the second organism or the original culture of the1,3-butanediol pathway intermediate producers can be depleted of thesemicrobial organisms by, for example, cell separation, and thensubsequent addition of the second organism to the fermentation broth canbe utilized to produce the final product without intermediatepurification steps.

In other embodiments, the non-naturally occurring microbial organismsand methods of the invention can be assembled in a wide variety ofsubpathways to achieve biosynthesis of, for example, 1,3-butanediol. Inthese embodiments, biosynthetic pathways for a desired product of theinvention can be segregated into different microbial organisms, and thedifferent microbial organisms can be co-cultured to produce the finalproduct. In such a biosynthetic scheme, the product of one microbialorganism is the substrate for a second microbial organism until thefinal product is synthesized. For example, the biosynthesis of1,3-butanediol can be accomplished by constructing a microbial organismthat contains biosynthetic pathways for conversion of one pathwayintermediate to another pathway intermediate or the product.Alternatively, 1,3-butanediol also can be biosynthetically produced frommicrobial organisms through co-culture or co-fermentation using twoorganisms in the same vessel, where the first microbial organismproduces a 1,3-butanediol intermediate and the second microbial organismconverts the intermediate to 1,3-butanediol.

Given the teachings and guidance provided herein, those skilled in theart will understand that a wide variety of combinations and permutationsexist for the non-naturally occurring microbial organisms and methods ofthe invention together with other microbial organisms, with theco-culture of other non-naturally occurring microbial organisms havingsubpathways and with combinations of other chemical and/or biochemicalprocedures well known in the art to produce 1,3-butanediol.

Sources of encoding nucleic acids for a 1,3-butanediol pathway enzyme orprotein can include, for example, any species where the encoded geneproduct is capable of catalyzing the referenced reaction. Such speciesinclude both prokaryotic and eukaryotic organisms including, but notlimited to, bacteria, including archaea and eubacteria, and eukaryotes,including yeast, plant, algae, insect, animal, and mammal, includinghuman. Exemplary species for such sources include, for example,Escherichia coli, as well as other exemplary species disclosed herein oravailable as source organisms for corresponding genes. However, with thecomplete genome sequence available for now more than 550 species (withmore than half of these available on public databases such as the NCBI),including 395 microorganism genomes and a variety of yeast, fungi,plant, and mammalian genomes, the identification of genes encoding therequisite 1,3-butanediol biosynthetic activity for one or more genes inrelated or distant species, including for example, homologues,orthologs, paralogs and nonorthologous gene displacements of knowngenes, and the interchange of genetic alterations between organisms isroutine and well known in the art. Accordingly, the metabolicalterations allowing biosynthesis of 1,3-butanediol described hereinwith reference to a particular organism such as E. coli can be readilyapplied to other microorganisms, including prokaryotic and eukaryoticorganisms alike. Given the teachings and guidance provided herein, thoseskilled in the art will know that a metabolic alteration exemplified inone organism can be applied equally to other organisms.

In some instances, such as when an alternative 1,3-butanediolbiosynthetic pathway exists in an unrelated species, 1,3-butanediolbiosynthesis can be conferred onto the host species by, for example,exogenous expression of a paralog or paralogs from the unrelated speciesthat catalyzes a similar, yet non-identical metabolic reaction toreplace the referenced reaction. Because certain differences amongmetabolic networks exist between different organisms, those skilled inthe art will understand that the actual gene usage between differentorganisms may differ. However, given the teachings and guidance providedherein, those skilled in the art also will understand that the teachingsand methods of the invention can be applied to all microbial organismsusing the cognate metabolic alterations to those exemplified herein toconstruct a microbial organism in a species of interest that willsynthesize 1,3-butanediol.

Methods for constructing and testing the expression levels of anon-naturally occurring 1,3-butanediol-producing host can be performed,for example, by recombinant and detection methods well known in the art.Such methods can be found described in, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring HarborLaboratory, New York (2001); and Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1999).

Exogenous nucleic acid sequences involved in a pathway for production of1,3-butanediol can be introduced stably or transiently into a host cellusing techniques well known in the art including, but not limited to,conjugation, electroporation, chemical transformation, transduction,transfection, and ultrasound transformation. For exogenous expression inE. coli or other prokaryotic cells, some nucleic acid sequences in thegenes or cDNAs of eukaryotic nucleic acids can encode targeting signalssuch as an N-terminal mitochondrial or other targeting signal, which canbe removed before transformation into prokaryotic host cells, ifdesired. For example, removal of a mitochondrial leader sequence led toincreased expression in E. coli (Hoffmeister et al., J. Biol. Chem.280:4329-4338 (2005)). For exogenous expression in yeast or othereukaryotic cells, genes can be expressed in the cytosol without theaddition of leader sequence, or can be targeted to mitochondrion orother organelles, or targeted for secretion, by the addition of asuitable targeting sequence such as a mitochondrial targeting orsecretion signal suitable for the host cells. Thus, it is understoodthat appropriate modifications to a nucleic acid sequence to remove orinclude a targeting sequence can be incorporated into an exogenousnucleic acid sequence to impart desirable properties. Furthermore, genescan be subjected to codon optimization with techniques well known in theart to achieve optimized expression of the proteins.

An expression vector or vectors can be constructed to include one ormore 1,3-butanediol biosynthetic pathway encoding nucleic acids asexemplified herein operably linked to expression control sequencesfunctional in the host organism. Expression vectors applicable for usein the microbial host organisms of the invention include, for example,plasmids, phage vectors, viral vectors, episomes and artificialchromosomes, including vectors and selection sequences or markersoperable for stable integration into a host chromosome. Additionally,the expression vectors can include one or more selectable marker genesand appropriate expression control sequences. Selectable marker genesalso can be included that, for example, provide resistance toantibiotics or toxins, complement auxotrophic deficiencies, or supplycritical nutrients not in the culture media. Expression controlsequences can include constitutive and inducible promoters,transcription enhancers, transcription terminators, and the like whichare well known in the art. When two or more exogenous encoding nucleicacids are to be co-expressed, both nucleic acids can be inserted, forexample, into a single expression vector or in separate expressionvectors. For single vector expression, the encoding nucleic acids can beoperationally linked to one common expression control sequence or linkedto different expression control sequences, such as one induciblepromoter and one constitutive promoter. The transformation of exogenousnucleic acid sequences involved in a metabolic or synthetic pathway canbe confirmed using methods well known in the art. Such methods include,for example, nucleic acid analysis such as Northern blots or polymerasechain reaction (PCR) amplification of mRNA, or immunoblotting forexpression of gene products, or other suitable analytical methods totest the expression of an introduced nucleic acid sequence or itscorresponding gene product. It is understood by those skilled in the artthat the exogenous nucleic acid is expressed in a sufficient amount toproduce the desired product, and it is further understood thatexpression levels can be optimized to obtain sufficient expression usingmethods well known in the art and as disclosed herein.

In some embodiments, the present invention provides a method forproducing 1,3-BDO that includes culturing a non-naturally occurringmicrobial organism having a 1,3-BDO pathway having at least oneexogenous nucleic acid encoding a 1,3-BDO pathway enzyme or proteinexpressed in a sufficient amount to produce 1,3-BDO, under conditionsand for a sufficient period of time to produce 1,3-BDO. In someembodiments, the 1,3-BDO pathway includes Methanol methyltransferase(MtaB), Corrinoid protein (MtaC), Methyltetrahydrofolate:corrinoidprotein methyltransferase (MtaA), Methyltetrahydrofolate:corrinoidprotein methyltransferase (AcsE), Corrinoid iron-sulfur protein (AcsD),Nickel-protein assembly protein (AcsF & CooC), Ferredoxin (Orf7),Acetyl-CoA synthase (AcsB & AcsC), Carbon monoxide dehydrogenase (AcsA),Hydrogenase (Hyd), Acetoacetyl-CoA thiolase (AtoB), Acetoacetyl-CoAreductase (CoA-dependent, aldehyde forming), 3-oxobutyraldehydereductase (ketone reducing), 3-hydroxybutyraldehyde reductase,Acetoacetyl-CoA reductase (CoA-dependent, alcohol forming),3-oxobutyraldehyde reductase (aldehyde reducing), 4-hydroxy,2-butanonereductase, Acetoacetyl-CoA reductase (ketone reducing),3-hydroxybutyryl-CoA reductase (aldehyde forming), 3-hydroxybutyryl-CoAreductase (alcohol forming), 3-hydroxybutyryl-CoA transferase,3-hydroxybutyryl-CoA hydrolase, 3-hydroxybutyryl-CoA synthetase,3-hydroxybutyrate dehydrogenase, 3-hydroxybutyrate reductase,acetoacetyl-CoA transferase, acetoacetyl-CoA hydrolase, acetoacetyl-CoAsynthetase, or acetoacetate reductase.

In other embodiments, the present invention provides a method forproducing 1,3-BDO that includes culturing a non-naturally occurringmicrobial organism having a 1,3-BDO pathway having at least oneexogenous nucleic acid encoding a 1,3-BDO pathway enzyme or proteinexpressed in a sufficient amount to produce 1,3-BDO, under conditionsand for a sufficient period of time to produce 1,3-BDO. The 1,3-BDOpathway includes Formate dehydrogenase, Formyltetrahydrofolatesynthetase, Methenyltetrahydrofolate cyclohydrolase,Methylenetetrahydrofolate dehydrogenase, Methylenetetrahydrofolatereductase, Methyltetrahydrofolate:corrinoid protein methyltransferase(AcsE), Corrinoid iron-sulfur protein (AcsD), Nickel-protein assemblyprotein (AcsF & CooC), Ferredoxin (Orf7), Acetyl-CoA synthase (AcsB &AcsC), Carbon monoxide dehydrogenase (AcsA), Hydrogenase (Hyd),Acetoacetyl-CoA thiolase (AtoB), Acetoacetyl-CoA reductase(CoA-dependent, aldehyde forming), 3-oxobutyraldehyde reductase (ketonereducing), 3-hydroxybutyraldehyde reductase, Acetoacetyl-CoA reductase(CoA-dependent, alcohol forming), 3-oxobutyraldehyde reductase (aldehydereducing), 4-hydroxy,2-butanone reductase, Acetoacetyl-CoA reductase(ketone reducing), 3-hydroxybutyryl-CoA reductase (aldehyde forming),3-hydroxybutyryl-CoA reductase (alcohol forming), 3-hydroxybutyryl-CoAtransferase, 3-hydroxybutyryl-CoA hydrolase, 3-hydroxybutyryl-CoAsynthetase, 3-hydroxybutyrate dehydrogenase, 3-hydroxybutyratereductase, acetoacetyl-CoA transferase, acetoacetyl-CoA hydrolase,acetoacetyl-CoA synthetase, or acetoacetate reductase.

In some embodiments, culturing the non-naturally occurring microbialorganism includes culturing under conditions and for a sufficient periodof time to produce 1,3-BDO. In some embodiments, culturing is performedin a substantially anaerobic culture medium. In some embodiments, atleast one exogenous nucleic acid of the microbial organism is aheterologous nucleic acid. As described above, the culturednon-naturally occurring microbial organisms can have any number ofexogenous nucleic acids in a 1,3-BDO pathway including 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, that is up to all the nucleicacids encoding a 1,3-BDO pathway. Non-naturally occurring microbialorganisms of the invention can utilize a carbon feedstock selectedfrom 1) methanol and CO, 2) methanol, CO₂, and H₂, 3) methanol, CO, CO₂,and H₂, 4) methanol and synthesis gas comprising CO and H₂, 5) methanoland synthesis gas comprising CO, CO₂, and H₂, 6) one or morecarbohydrates, 7) methanol and one or more carbohydrates, and 8)methanol, in some embodiments or a carbon feedstock selected from 1) CO,2) CO₂ and H₂, 3) CO, CO₂, and H₂, 4) synthesis gas comprising CO andH₂, 5) synthesis gas comprising CO, CO₂, and H₂, and 6) one or morecarbohydrates, in other embodiments.

Suitable purification and/or assays to test for the production of1,3-butanediol can be performed using well known methods. Suitablereplicates such as triplicate cultures can be grown for each engineeredstrain to be tested. For example, product and byproduct formation in theengineered production host can be monitored. The final product andintermediates, and other organic compounds, can be analyzed by methodssuch as HPLC (High Performance Liquid Chromatography), GC-MS (GasChromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-MassSpectroscopy) or other suitable analytical methods using routineprocedures well known in the art. The release of product in thefermentation broth can also be tested with the culture supernatant.Byproducts and residual glucose can be quantified by HPLC using, forexample, a refractive index detector for glucose and alcohols, and a UVdetector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779(2005)), or other suitable assay and detection methods well known in theart. The individual enzyme or protein activities from the exogenous DNAsequences can also be assayed using methods well known in the art.

The 1,3-butanediol can be separated from other components in the cultureusing a variety of methods well known in the art. Such separationmethods include, for example, extraction procedures as well as methodsthat include continuous liquid-liquid extraction, pervaporation,membrane filtration, membrane separation, reverse osmosis,electrodialysis, distillation, crystallization, centrifugation,extractive filtration, ion exchange chromatography, size exclusionchromatography, adsorption chromatography, and ultrafiltration. All ofthe above methods are well known in the art.

Any of the non-naturally occurring microbial organisms described hereincan be cultured to produce and/or secrete the biosynthetic products ofthe invention. For example, the 1,3-butanediol producers can be culturedfor the biosynthetic production of 1,3-butanediol.

For the production of 1,3-butanediol, the recombinant strains arecultured in a medium with carbon source and other essential nutrients.It is highly desirable to maintain anaerobic conditions in the fermenterto reduce the cost of the overall process. Such conditions can beobtained, for example, by first sparging the medium with nitrogen andthen sealing the flasks with a septum and crimp-cap. For strains wheregrowth is not observed anaerobically, microaerobic conditions can beapplied by perforating the septum with a small hole for limitedaeration. Exemplary anaerobic conditions have been described previouslyand are well-known in the art. Exemplary aerobic and anaerobicconditions are described, for example, in U.S. patent application Ser.No. 11/891,602, filed Aug. 10, 2007. Fermentations can be performed in abatch, fed-batch or continuous manner, as disclosed herein.

If desired, the pH of the medium can be maintained at a desired pH, inparticular neutral pH, such as a pH of around 7 by addition of a base,such as NaOH or other bases, or acid, as needed to maintain the culturemedium at a desirable pH. The growth rate can be determined by measuringoptical density using a spectrophotometer (600 nm), and the glucoseuptake rate by monitoring carbon source depletion over time.

Accordingly, given the teachings and guidance provided herein, thoseskilled in the art will understand that a non-naturally occurringmicrobial organism can be produced that secretes the biosynthesizedcompounds of the invention when grown on a carbon source such as syngas,methanol, or combinations of CO, CO₂, hydrogen, and the like. Suchcompounds include, for example, 1,3-butanediol and any of theintermediate metabolites in the 1,3-butanediol pathway. All that isrequired is to engineer in one or more of the required enzyme or proteinactivities to achieve biosynthesis of the desired compound orintermediate including, for example, inclusion of some or all of the1,3-butanediol biosynthetic pathways.

The non-naturally occurring microbial organisms of the invention areconstructed using methods well known in the art as exemplified herein toexogenously express at least one nucleic acid encoding a 1,3-butanediolpathway enzyme or protein in sufficient amounts to produce1,3-butanediol. It is understood that the microbial organisms of theinvention are cultured under conditions sufficient to produce1,3-butanediol. Following the teachings and guidance provided herein,the non-naturally occurring microbial organisms of the invention canachieve biosynthesis of 1,3-butanediol resulting in intracellularconcentrations between about 0.1-200 mM or more. Generally, theintracellular concentration of 1,3-butanediol is between about 3-150 mM,particularly between about 5-125 mM and more particularly between about8-100 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, or more.Intracellular concentrations between and above each of these exemplaryranges also can be achieved from the non-naturally occurring microbialorganisms of the invention.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described herein and are described, for example, in U.S. publication2009/0047719, filed Aug. 10, 2007. Any of these conditions can beemployed with the non-naturally occurring microbial organisms as well asother anaerobic conditions well known in the art. Under such anaerobicconditions, the 1,3-butanediol producers can synthesize 1,3-butanediolat intracellular concentrations of 5-10 mM or more as well as all otherconcentrations exemplified herein. It is understood that, even thoughthe above description refers to intracellular concentrations,1,3-butanediol producing microbial organisms can produce 1,3-butanediolintracellularly and/or secrete the product into the culture medium.

In addition to the culturing and fermentation conditions disclosedherein, growth condition for achieving biosynthesis of 1,3-butanediolcan include the addition of an osmoprotectant to the culturingconditions. In certain embodiments, the non-naturally occurringmicrobial organisms of the invention can be sustained, cultured orfermented as described herein in the presence of an osmoprotectant.Briefly, an osmoprotectant refers to a compound that acts as an osmolyteand helps a microbial organism as described herein survive osmoticstress. Osmoprotectants include, but are not limited to, betaines, aminoacids, and the sugar trehalose. Non-limiting examples of such areglycine betaine, praline betaine, dimethylthetin,dimethylslfonioproprionate, 3-dimethylsulfonio-2-methylproprionate,pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine andectoine. In one aspect, the osmoprotectant is glycine betaine. It isunderstood to one of ordinary skill in the art that the amount and typeof osmoprotectant suitable for protecting a microbial organism describedherein from osmotic stress will depend on the microbial organism used.The amount of osmoprotectant in the culturing conditions can be, forexample, no more than about 0.1 mM, no more than about 0.5 mM, no morethan about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM,no more than about 2.5 mM, no more than about 3.0 mM, no more than about5.0 mM, no more than about 7.0 mM, no more than about 10 mM, no morethan about 50 mM, no more than about 100 mM or no more than about 500mM.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described herein, particularly useful yields of thebiosynthetic products of the invention can be obtained under anaerobicor substantially anaerobic culture conditions

As described herein, one exemplary growth condition for achievingbiosynthesis of 1,3-butanediol includes anaerobic culture orfermentation conditions. In certain embodiments, the non-naturallyoccurring microbial organisms of the invention can be sustained,cultured or fermented under anaerobic or substantially anaerobicconditions. Briefly, anaerobic conditions refer to an environment devoidof oxygen. Substantially anaerobic conditions include, for example, aculture, batch fermentation or continuous fermentation such that thedissolved oxygen concentration in the medium remains between 0 and 10%of saturation. Substantially anaerobic conditions also includes growingor resting cells in liquid medium or on solid agar inside a sealedchamber maintained with an atmosphere of less than 1% oxygen. Thepercent of oxygen can be maintained by, for example, sparging theculture with an N₂/CO₂ mixture or other suitable non-oxygen gas orgases.

The culture conditions described herein can be scaled up and growncontinuously for manufacturing of 1,3-butanediol. Exemplary growthprocedures include, for example, fed-batch fermentation and batchseparation; fed-batch fermentation and continuous separation, orcontinuous fermentation and continuous separation. All of theseprocesses are well known in the art. Fermentation procedures areparticularly useful for the biosynthetic production of commercialquantities of 1,3-butanediol. Generally, and as with non-continuousculture procedures, the continuous and/or near-continuous production of1,3-butanediol will include culturing a non-naturally occurring1,3-butanediol producing organism of the invention in sufficientnutrients and medium to sustain and/or nearly sustain growth in anexponential phase. Continuous culture under such conditions can include,for example, growth for 1 day, 2, 3, 4, 5, 6 or 7 days or more.Additionally, continuous culture can include longer time periods of 1week, 2, 3, 4 or 5 or more weeks and up to several months.Alternatively, organisms of the invention can be cultured for hours, ifsuitable for a particular application. It is to be understood that thecontinuous and/or near-continuous culture conditions also can includeall time intervals in between these exemplary periods. It is furtherunderstood that the time of culturing the microbial organism of theinvention is for a sufficient period of time to produce a sufficientamount of product for a desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentationfor the biosynthetic production of 1,3-butanediol can be utilized in,for example, fed-batch fermentation and batch separation; fed-batchfermentation and continuous separation, or continuous fermentation andcontinuous separation. Examples of batch and continuous fermentationprocedures are well known in the art.

In addition to the above fermentation procedures using the1,3-butanediol producers of the invention for continuous production ofsubstantial quantities of 1,3-butanediol, the 1,3-butanediol producersalso can be, for example, simultaneously subjected to chemical synthesisprocedures to convert the product to other compounds or the product canbe separated from the fermentation culture and sequentially subjected tochemical conversion to convert the product to other compounds, ifdesired.

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. patent publications US 2002/0012939, US 2003/0224363,US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 andUS 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allowsreliable predictions of the effects on cell growth of shifting themetabolism towards more efficient production of 1,3-butanediol.

One computational method for identifying and designing metabolicalterations favoring biosynthesis of a desired product is the OptKnockcomputational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657(2003)). OptKnock is a metabolic modeling and simulation program thatsuggests gene deletion or disruption strategies that result ingenetically stable microorganisms which overproduce the target product.Specifically, the framework examines the complete metabolic and/orbiochemical network of a microorganism in order to suggest geneticmanipulations that force the desired biochemical to become an obligatorybyproduct of cell growth. By coupling biochemical production with cellgrowth through strategically placed gene deletions or other functionalgene disruption, the growth selection pressures imposed on theengineered strains after long periods of time in a bioreactor lead toimprovements in performance as a result of the compulsory growth-coupledbiochemical production. Lastly, when gene deletions are constructedthere is a negligible possibility of the designed strains reverting totheir wild-type states because the genes selected by OptKnock are to becompletely removed from the genome. Therefore, this computationalmethodology can be used to either identify alternative pathways thatlead to biosynthesis of a desired product or used in connection with thenon-naturally occurring microbial organisms for further optimization ofbiosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or deletions. OptKnockcomputational framework allows the construction of model formulationsthat allow an effective query of the performance limits of metabolicnetworks and provides methods for solving the resulting mixed-integerlinear programming problems. The metabolic modeling and simulationmethods referred to herein as OptKnock are described in, for example,U.S. publication 2002/0168654, filed Jan. 10, 2002, in InternationalPatent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. publication2009/0047719, filed Aug. 10, 2007.

Another computational method for identifying and designing metabolicalterations favoring biosynthetic production of a product is a metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. publication2003/0233218, filed Jun. 14, 2002, and in International PatentApplication No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is acomputational system that can be used to produce a network model insilico and to simulate the flux of mass, energy or charge through thechemical reactions of a biological system to define a solution spacethat contains any and all possible functionalities of the chemicalreactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realitiesbecause biological systems are flexible and can reach the same result inmany different ways. Biological systems are designed throughevolutionary mechanisms that have been restricted by fundamentalconstraints that all living systems must face. Therefore,constraints-based modeling strategy embraces these general realities.Further, the ability to continuously impose further restrictions on anetwork model via the tightening of constraints results in a reductionin the size of the solution space, thereby enhancing the precision withwhich physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in theart can be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of adesired compound in host microbial organisms. Such metabolic modelingand simulation methods include, for example, the computational systemsexemplified above as SimPheny® and OptKnock. For illustration of theinvention, some methods are described herein with reference to theOptKnock computation framework for modeling and simulation. Thoseskilled in the art will know how to apply the identification, design andimplementation of the metabolic alterations using OptKnock to any ofsuch other metabolic modeling and simulation computational frameworksand methods well known in the art.

The methods described above will provide one set of metabolic reactionsto disrupt. Elimination of each reaction within the set or metabolicmodification can result in a desired product as an obligatory productduring the growth phase of the organism. Because the reactions areknown, a solution to the bilevel OptKnock problem also will provide theassociated gene or genes encoding one or more enzymes that catalyze eachreaction within the set of reactions. Identification of a set ofreactions and their corresponding genes encoding the enzymesparticipating in each reaction is generally an automated process,accomplished through correlation of the reactions with a reactiondatabase having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in orderto achieve production of a desired product are implemented in the targetcell or organism by functional disruption of at least one gene encodingeach metabolic reaction within the set. One particularly useful means toachieve functional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the coupling of a product are desiredor when genetic reversion is less likely to occur.

To identify additional productive solutions to the above describedbilevel OptKnock problem which lead to further sets of reactions todisrupt or metabolic modifications that can result in the biosynthesis,including growth-coupled biosynthesis of a desired product, anoptimization method, termed integer cuts, can be implemented. Thismethod proceeds by iteratively solving the OptKnock problem exemplifiedabove with the incorporation of an additional constraint referred to asan integer cut at each iteration. Integer cut constraints effectivelyprevent the solution procedure from choosing the exact same set ofreactions identified in any previous iteration that obligatorily couplesproduct biosynthesis to growth. For example, if a previously identifiedgrowth-coupled metabolic modification specifies reactions 1, 2, and 3for disruption, then the following constraint prevents the samereactions from being simultaneously considered in subsequent solutions.The integer cut method is well known in the art and can be founddescribed in, for example, Burgard et al., Biotechnol. Prog. 17:791-797(2001). As with all methods described herein with reference to their usein combination with the OptKnock computational framework for metabolicmodeling and simulation, the integer cut method of reducing redundancyin iterative computational analysis also can be applied with othercomputational frameworks well known in the art including, for example,SimPheny®.

The methods exemplified herein allow the construction of cells andorganisms that biosynthetically produce a desired product, including theobligatory coupling of production of a target biochemical product togrowth of the cell or organism engineered to harbor the identifiedgenetic alterations. Therefore, the computational methods describedherein allow the identification and implementation of metabolicmodifications that are identified by an in silico method selected fromOptKnock or SimPheny®. The set of metabolic modifications can include,for example, addition of one or more biosynthetic pathway enzymes and/orfunctional disruption of one or more metabolic reactions including, forexample, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on thepremise that mutant microbial networks can be evolved towards theircomputationally predicted maximum-growth phenotypes when subjected tolong periods of growth selection. In other words, the approach leveragesan organism's ability to self-optimize under selective pressures. TheOptKnock framework allows for the exhaustive enumeration of genedeletion combinations that force a coupling between biochemicalproduction and cell growth based on network stoichiometry. Theidentification of optimal gene/reaction knockouts requires the solutionof a bilevel optimization problem that chooses the set of activereactions such that an optimal growth solution for the resulting networkoverproduces the biochemical of interest (Burgard et al., Biotechnol.Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employedto identify essential genes for metabolic pathways as exemplifiedpreviously and described in, for example, U.S. patent publications US2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No.7,127,379. As disclosed herein, the OptKnock mathematical framework canbe applied to pinpoint gene deletions leading to the growth-coupledproduction of a desired product. Further, the solution of the bilevelOptKnock problem provides only one set of deletions. To enumerate allmeaningful solutions, that is, all sets of knockouts leading togrowth-coupled production formation, an optimization technique, termedinteger cuts, can be implemented. This entails iteratively solving theOptKnock problem with the incorporation of an additional constraintreferred to as an integer cut at each iteration, as discussed above.

As disclosed herein, a nucleic acid encoding a desired activity of a1,3-butanediol pathway can be introduced into a host organism. In somecases, it can be desirable to modify an activity of a 1,3-butanediolpathway enzyme or protein to increase production of 1,3-butanediol. Forexample, known mutations that increase the activity of a protein orenzyme can be introduced into an encoding nucleic acid molecule.Additionally, optimization methods can be applied to increase theactivity of an enzyme or protein and/or decrease an inhibitory activity,for example, decrease the activity of a negative regulator.

One such optimization method is directed evolution. Directed evolutionis a powerful approach that involves the introduction of mutationstargeted to a specific gene in order to improve and/or alter theproperties of an enzyme. Improved and/or altered enzymes can beidentified through the development and implementation of sensitivehigh-throughput screening assays that allow the automated screening ofmany enzyme variants (for example, >104). Iterative rounds ofmutagenesis and screening typically are performed to afford an enzymewith optimized properties. Computational algorithms that can help toidentify areas of the gene for mutagenesis also have been developed andcan significantly reduce the number of enzyme variants that need to begenerated and screened. Numerous directed evolution technologies havebeen developed (for reviews, see Hibbert et al., Biomol. Eng 22:11-19(2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical andbiotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press;Otten and Quax. Biomol. Eng 22:1-9 (2005); and Sen et al., Appl Biochem.Biotechnol 143:212-223 (2007)) to be effective at creating diversevariant libraries, and these methods have been successfully applied tothe improvement of a wide range of properties across many enzymeclasses. Enzyme characteristics that have been improved and/or alteredby directed evolution technologies include, for example:selectivity/specificity, for conversion of non-natural substrates;temperature stability, for robust high temperature processing; pHstability, for bioprocessing under lower or higher pH conditions;substrate or product tolerance, so that high product titers can beachieved; binding (K_(m)), including broadening substrate binding toinclude non-natural substrates; inhibition (K_(i)), to remove inhibitionby products, substrates, or key intermediates; activity (kcat), toincreases enzymatic reaction rates to achieve desired flux; expressionlevels, to increase protein yields and overall pathway flux; oxygenstability, for operation of air sensitive enzymes under aerobicconditions; and anaerobic activity, for operation of an aerobic enzymein the absence of oxygen.

A number of exemplary methods have been developed for the mutagenesisand diversification of genes to target desired properties of specificenzymes. Such methods are well known to those skilled in the art. Any ofthese can be used to alter and/or optimize the activity of a1,3-butanediol pathway enzyme or protein. Such methods include, but arenot limited to EpPCR, which introduces random point mutations byreducing the fidelity of DNA polymerase in PCR reactions (Pritchard etal., J Theor. Biol. 234:497-509 (2005)); Error-prone Rolling CircleAmplification (epRCA), which is similar to epPCR except a whole circularplasmid is used as the template and random 6-mers with exonucleaseresistant thiophosphate linkages on the last 2 nucleotides are used toamplify the plasmid followed by transformation into cells in which theplasmid is re-circularized at tandem repeats (Fujii et al., NucleicAcids Res. 32:e145 (2004); and Fujii et al., Nat. Protoc. 1:2493-2497(2006)); DNA or Family Shuffling, which typically involves digestion oftwo or more variant genes with nucleases such as Dnase I or EndoV togenerate a pool of random fragments that are reassembled by cycles ofannealing and extension in the presence of DNA polymerase to create alibrary of chimeric genes (Stemmer, Proc Natl Acad Sci USA91:10747-10751 (1994); and Stemmer, Nature 370:389-391 (1994));Staggered Extension (StEP), which entails template priming followed byrepeated cycles of 2 step PCR with denaturation and very short durationof annealing/extension (as short as 5 sec) (Zhao et al., Nat.Biotechnol. 16:258-261 (1998)); Random Priming Recombination (RPR), inwhich random sequence primers are used to generate many short DNAfragments complementary to different segments of the template (Shao etal., Nucleic Acids Res 26:681-683 (1998)).

Additional methods include Heteroduplex Recombination, in whichlinearized plasmid DNA is used to form heteroduplexes that are repairedby mismatch repair (Volkov et al, Nucleic Acids Res. 27:e18 (1999); andVolkov et al., Methods Enzymol. 328:456-463 (2000)); RandomChimeragenesis on Transient Templates (RACHITT), which employs Dnase Ifragmentation and size fractionation of single stranded DNA (ssDNA)(Coco et al., Nat. Biotechnol. 19:354-359 (2001)); Recombined Extensionon Truncated templates (RETT), which entails template switching ofunidirectionally growing strands from primers in the presence ofunidirectional ssDNA fragments used as a pool of templates (Lee et al.,J. Molec. Catalysis 26:119-129 (2003)); Degenerate Oligonucleotide GeneShuffling (DOGS), in which degenerate primers are used to controlrecombination between molecules; (Bergquist and Gibbs, Methods Mol.Biol. 352:191-204 (2007); Bergquist et al., Biomol. Eng 22:63-72 (2005);Gibbs et al., Gene 271:13-20 (2001)); Incremental Truncation for theCreation of Hybrid Enzymes (ITCHY), which creates a combinatoriallibrary with 1 base pair deletions of a gene or gene fragment ofinterest (Ostermeier et al., Proc. Natl. Acad. Sci. USA 96:3562-3567(1999); and Ostermeier et al., Nat. Biotechnol. 17:1205-1209 (1999));Thio-Incremental Truncation for the Creation of Hybrid Enzymes(THIO-ITCHY), which is similar to ITCHY except that phosphothioate dNTPsare used to generate truncations (Lutz et al., Nucleic Acids Res 29:E16(2001)); SCRATCHY, which combines two methods for recombining genes,ITCHY and DNA shuffling (Lutz et al., Proc. Natl. Acad. Sci. USA98:11248-11253 (2001)); Random Drift Mutagenesis (RNDM), in whichmutations made via epPCR are followed by screening/selection for thoseretaining usable activity (Bergquist et al., Biomol. Eng. 22:63-72(2005)); Sequence Saturation Mutagenesis (SeSaM), a random mutagenesismethod that generates a pool of random length fragments using randomincorporation of a phosphothioate nucleotide and cleavage, which is usedas a template to extend in the presence of “universal” bases such asinosine, and replication of an inosine-containing complement givesrandom base incorporation and, consequently, mutagenesis (Wong et al.,Biotechnol. J. 3:74-82 (2008); Wong et al., Nucleic Acids Res. 32:e26(2004); and Wong et al., Anal. Biochem. 341:187-189 (2005)); SyntheticShuffling, which uses overlapping oligonucleotides designed to encode“all genetic diversity in targets” and allows a very high diversity forthe shuffled progeny (Ness et al., Nat. Biotechnol. 20:1251-1255(2002)); Nucleotide Exchange and Excision Technology NexT, whichexploits a combination of dUTP incorporation followed by treatment withuracil DNA glycosylase and then piperidine to perform endpoint DNAfragmentation (Muller et al., Nucleic Acids Res. 33:e117 (2005)).

Further methods include Sequence Homology-Independent ProteinRecombination (SHIPREC), in which a linker is used to facilitate fusionbetween two distantly related or unrelated genes, and a range ofchimeras is generated between the two genes, resulting in libraries ofsingle-crossover hybrids (Sieber et al., Nat. Biotechnol. 19:456-460(2001)); Gene Site Saturation Mutagenesis™ (GSSM™), in which thestarting materials include a supercoiled double stranded DNA (dsDNA)plasmid containing an insert and two primers which are degenerate at thedesired site of mutations (Kretz et al., Methods Enzymol. 388:3-11(2004)); Combinatorial Cassette Mutagenesis (CCM), which involves theuse of short oligonucleotide cassettes to replace limited regions with alarge number of possible amino acid sequence alterations (Reidhaar-Olsonet al. Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al.Science 241:53-57 (1988)); Combinatorial Multiple Cassette Mutagenesis(CMCM), which is essentially similar to CCM and uses epPCR at highmutation rate to identify hot spots and hot regions and then extensionby CMCM to cover a defined region of protein sequence space (Reetz etal., Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001)); the MutatorStrains technique, in which conditional is mutator plasmids, utilizingthe mutD5 gene, which encodes a mutant subunit of DNA polymerase III, toallow increases of 20 to 4000-X in random and natural mutation frequencyduring selection and block accumulation of deleterious mutations whenselection is not required (Selifonova et al., Appl. Environ. Microbiol.67:3645-3649 (2001)); Low et al., J. Mol. Biol. 260:359-3680 (1996)).

Additional exemplary methods include Look-Through Mutagenesis (LTM),which is a multidimensional mutagenesis method that assesses andoptimizes combinatorial mutations of selected amino acids (Rajpal etal., Proc. Natl. Acad. Sci. USA 102:8466-8471 (2005)); Gene Reassembly,which is a DNA shuffling method that can be applied to multiple genes atone time or to create a large library of chimeras (multiple mutations)of a single gene (Tunable GeneReassembly™ (TGR™) Technology supplied byVerenium Corporation), in Silico Protein Design Automation (PDA), whichis an optimization algorithm that anchors the structurally definedprotein backbone possessing a particular fold, and searches sequencespace for amino acid substitutions that can stabilize the fold andoverall protein energetics, and generally works most effectively onproteins with known three-dimensional structures (Hayes et al., Proc.Natl. Acad. Sci. USA 99:15926-15931 (2002)); and Iterative SaturationMutagenesis (ISM), which involves using knowledge of structure/functionto choose a likely site for enzyme improvement, performing saturationmutagenesis at chosen site using a mutagenesis method such as StratageneQuikChange (Stratagene; San Diego Calif.), screening/selecting fordesired properties, and, using improved clone(s), starting over atanother site and continue repeating until a desired activity is achieved(Reetz et al., Nat. Protoc. 2:891-903 (2007); and Reetz et al., Angew.Chem. Int. Ed Engl. 45:7745-7751 (2006)).

Any of the aforementioned methods for mutagenesis can be used alone orin any combination. Additionally, any one or combination of the directedevolution methods can be used in conjunction with adaptive evolutiontechniques, as described herein.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoincluded within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

Example I Production of 1,3-BDO Using Methanol, CO, and/or CO₂ as CarbonFeedstock

This example shows how a non-naturally occurring organism can beconstructed to produce 1,3-BDO from methanol, CO, and/or CO₂ as thecarbon source.

The first step in the cloning and expression process is to express in E.coli the minimal set of genes (e.g., MtaA, MtaB, and MtaC) necessary toproduce Me-THF from methanol. These methyltransferase activities requireCoenzyme B₁₂ (cobalamin) as a cofactor. In Moorella thermoacetica, acascade of methyltransferase proteins mediate incorporation of methanolderived methyl groups into the acetyl-CoA synthase pathway. Recent work(Das et al., Proteins 67.1:167-176 (2007)) indicates that MtaABC areencoded by Moth_(—)1208-09 and Moth_(—)2346. These genes are cloned viaproof-reading PCR and linked together for expression in a high-copynumber vector such as pZE22-S under control of the repressible PA1-lacO1promoter (Lutz and Bujard, Nucleic Acids Res. 25.6:1203-1210 (1997)).Cloned genes are verified by PCR and or restriction enzyme mapping todemonstrate construction and insertion of the 3-gene set into theexpression vector. DNA sequencing of the presumptive clones is carriedout to confirm the expected sequences of each gene. Once confirmed, thefinal construct is expressed in E. coli K-12 (MG1655) cells by additionof IPTG inducer between 0.05 and 1 mM final concentration. Expression ofthe cloned genes is monitored using SDS-PAGE of whole cell extracts. Tooptimize levels of soluble vs. pellet (potentially inclusion bodyorigin) protein, the affect of titration of the promoter on these levelscan be examined. If no acceptable expression is obtained, higher orlower copy number vectors or variants in promoter strength are tested.

To determine if expression of the MtaABC proteins from M. thermoaceticaconfers upon E. coli the ability to transfer methyl groups from methanolto tetrahydrofolate (THF) the recombinant strain is fed methanol atvarious concentrations. Activity of the methyltransferase system isassayed anaerobically as described for vanillate as a methyl source inM. thermoacetica (Naidu and Ragsdale, J. Bacteriol. 183.11:3276-3281(2001)) or for Methanosarcina barkeri methanol methyltransferase (Saueret al., Eur. J. Biochem. 243.3:670-677 (1997); Tallant et al., J. Biol.Chem. 276.6:4485-4493 (2001); Tallant and Krzycki, J. Bacteriol.179.22:6902-6911 (1997); Tallant and Krzycki, J. Bacteriol.178.5:1295-1301 (1996)). For a positive control, M. thermoacetica cellsare cultured in parallel and assayed anaerobically to confirm endogenousmethyltransferase activity. Demonstration of dependence on exogenouslyadded coenzyme B₁₂ confirms methanol:corrinoid methyltransferaseactivity in E. coli.

Once methyltransferase expression is achieved, further work is performedtowards optimizing the expression. Titrating the promoter in theexpression vector enables the testing of a range of expression levels.This is then used as a guide towards the expression needed insingle-copy, or allows the determination of whether or not a single-copyof these genes allows sufficient expression. If so, themethyltransferase genes are integrated into the chromosome as a single,synthetic operon. This entails targeted integration using RecET-based‘recombineering’ (Angrand et al., Nucleic Acids Res. 27.17:e16 (1999);Muyrers et al., Nucleic Acids Res. 27.6:1555-1557 (1999); Zhang et al.,Nat. Genet. 20.2:123-128 (1998)). A potential issue with RecET-basedintegration of a cassette and removal of a FRT or loxP-boundedselectable marker by FLP or Cre is the production of a recombinationscar at each integration site. While problems caused by this can beminimized by a number of methods, other means that do not leave genomicscars are available. The standard alternative is to introduce thedesired genes using integrative ‘suicide’ plasmids coupled tocounter-selection such as that allowed by the Bacillus sacB gene (Linket al., J. Bacteriol. 179.20:6228-6237 (1997)); in this way, markerlessand scar less insertions at any location in the E. coli chromosome canbe generated. The final goal is a strain of E. coli K-12 expressingmethanol:corrinoid methyltransferase activity under an induciblepromoter and in single copy (chromosomally integrated).

Using standard PCR methods, entire ACS/CODH operons are assembled intolow or medium copy number vectors such as pZA33-S (P15A-based) orpZS13-S (pSC101-based). As described for the methyltransferase genes,the structure and sequence of the cloned genes are confirmed. Expressionis monitored via protein gel electrophoresis of whole-cell lysates grownunder strictly anaerobic conditions with the requisite metals (Ni, Zn,Fe) and coenzyme B₁₂ provided. As necessary, the gene cluster ismodified for E. coli expression by identification and removal of anyapparent terminators and introduction of consensus ribosomal bindingsites chosen from sites known to be effective in E. coli (Barrick etal., Nucleic Acids Res. 22.7:1287-1295 (1994); Ringquist et al. Mol.Microbiol. 6.9:1219-1229). However, each gene cluster is cloned andexpressed in a manner parallel to its native structure and expression.This helps ensure the desired stoichiometry between the various geneproducts—most of which interact with each other. Once satisfactoryexpression of the CODH/ACS gene cluster under anaerobic conditions isachieved, the ability of cells expressing these genes to fix CO and/orCO₂ into cellular carbon is assayed. Initial conditions employ strictlyanaerobically grown cells provided with exogenous glucose as a carbonand energy source via substrate-level phosphorylation or anaerobicrespiration with nitrate as an electron acceptor. Additionally,exogenously provided CH₃-THF can be added to the medium.

The ACS/CODH genes are cloned and expressed in cells also expressing themethanol-methyltransferase system. This can be achieved by introductionof compatible plasmids expressing ACS/CODH into MTR-expressing cells.For added long-term stability, the ACS/CODH and MTR genes can also beintegrated into the chromosome. After strains of E. coli capable ofutilizing methanol to produce Me-THF and of expressing active CODH/ACSgene are made, they are assayed for the ability to utilize both methanoland syngas for incorporation into acetyl-CoA, acetate, and cell mass.Initial conditions employ strictly anaerobically grown cells providedwith exogenous glucose as a carbon and energy source. Alternatively, orin addition to glucose, nitrate can be added to the fermentation brothto serve as an electron acceptor and initiator of growth. Anaerobicgrowth of E. coli on fatty acids, which are ultimately metabolized toacetyl-CoA, has been demonstrated in the presence of nitrate (Campbellet al., Mol. Microbiol. 47.3:793-805 (2003)). Oxygen can also beprovided as long as its intracellular levels are maintained below anyinhibition threshold of the engineered enzymes. ¹³C-labeled methanol,¹³C-labeled bicarbonate or ¹³C-labeled CO are provided to the cells andanalytical mass spectrometry is employed to measure incorporation of thelabeled carbon into acetate and cell mass (e.g., proteinogenic aminoacids).

An alternative or supplement to engineering the methanolmethyltransferase system involves engineering the methyl branch of theWood-Ljungdahl pathway to supply ACS/CODH with the methyl group. WhileE. coli possesses genes encoding enzymes capable of carrying out some ofthe necessary activities (fdh, metF, folD), it has been indicated thatthe methyl branch enzymes from acetogens may have significantly higher(50-100×) specific activities than those from non-acetogens (Morton etal., Genetics and molecular biology of anaerobic bacteria SpringerVerlag, New York). The M. thermoacetica versions include formatedehydrogenase (fdhA, Moth_(—)2312-Moth_(—)2313 alpha, Moth_(—)2314beta), formyl-tetrahydrofolate (THF) synthetase (Moth_(—)0109),methenyl-THF cyclohydrolase/methylene-THF dehydrogenase (folD,Moth_(—)1516), methylene-THF reductase (metF, Moth_(—)1191), andmethyltransferase (acsE, Moth_(—)1197). With the exception of themethyltransferase (acsE, Moth_(—)1197) that will be cloned as part ofthe CODH/ACS cluster, these genes are cloned and combined into anexpression vector designed to express these as a set. Cloning andexpression of the methyl branch genes will be undertaken as describedabove. Initially, a high or medium copy number vector will be chosen(using ColE1 or P15A replicons). These genes can also be integrated intothe E. coli chromosome.

The pyruvate ferredoxin oxidoreductase genes from M. thermoacetica, D.africanus, and E. coli can be cloned and expressed in strains exhibitingACS/CODH activities. Conditions, promoters, etc., are described above.Given the large size of the PFOR genes and oxygen sensitivity of thecorresponding enzymes, tests can be performed using low or single-copyplasmid vectors or single-copy chromosomal integrations. Activity assaysdescribed in ref. (Furdui and Ragsdale, J. Biol. Chem.275.37:28494-28499 (2000)) can be applied to demonstrate activity. Inaddition, demonstration of growth on the gaseous carbon sources andmethanol in the absence of an external electron acceptor will providefurther evidence for PFOR activity in vivo.

The endogenous hydrogen-utilizing hydrogenase activity of the hostorganism can be tested by growing the cells as described above in thepresence and absence of hydrogen. If a dramatic shift towards theformation of more reduced products during fermentation is observed(e.g., increased ethanol as opposed to acetate), this indicates thatendogenous hydrogenase activity is sufficiently active. In this case, noheterologous hydrogenases are cloned and expressed. If the nativeenzymes do not have sufficient activity or reduce the needed acceptor,the genes encoding an individual hydrogenase complex can be cloned andexpressed in strains exhibiting ACS/CODH activities. Conditions,promoters, etc., are described above.

The normative genes needed for 1,3-butanediol synthesis are cloned onexpression plasmids as described previously. The host strain alsoexpresses methanol methyltransferase activity, CODH/ACS activity, andpossibly PFOR and hydrogenase activities. At this point, these(CODH/ACS, etc.) genes can be integrated into the genome and expressedfrom promoters that can be used constitutively or with inducers (i.e.,PA1-lacO1 is inducible in cells containing lad or is otherwiseconstitutive). Once expression and yields of 1,3-BDO are optimized, thebase strain can be further modified by integration of a single copy ofthese genes at a neutral locus. Given the relatively limited number ofenzymes (at minimum, 3, and at most, 4), one can construct an artificialoperon encoding the required genes. This operon can be introduced usingintegrative plasmids and is coupled to counter-selection methods such asthat allowed by the Bacillus sacB gene (Link et al., J. Bacteriol.179.20:6228-6237 (1997)). In this way, markerless and scar lessinsertions at any location in the E. coli chromosome can be generated.Optimization involves altering gene order as well as ribosomal bindingsites and promoters.

To over express any native genes, for example, the native atoB (b2224)gene of E. coli which can serve as an alternative to the C.acetobutylicum acetyl-coenzyme A [CoA] acetyltransferase required for1,3-butanediol production, RecET-based methods are applied to integratea stronger upstream promoter. In the case of atoB, this gene is the lastin an operon and the next gene downstream (yfaP) is both non-essentialand in the opposite orientation. Therefore, polarity should not be anissue. A cassette containing a selectable marker such as spectinomycinresistance or chloramphenicol resistance flanked by FRT or loxP sites isused to select for introduction of a strong constitutive promoter (e.g.,pL). Once the correct clone is obtained and validated, using qRT-PCR,FLP or Cre expression is used to select for removal of the FRT- orloxP-bounded marker.

Example II Cloning and Expression of Moorella Thermoacetica ACS/CODHEncoding Genes

This example describes the creation of E. coli plasmids that express theM. thermoacetica ACS/CODH operon genes including those used for CODH,ACS, methyltransferase, and the corrinoid iron-sulfur protein. Thisexample further describes the expression these in E. coli resulting inobservable CO oxidation activity, methyltransferase activity, andcorrinoid iron-sulfur protein activity. Finally, this exampledemonstrates that E. coli tolerates high CO concentrations, and may evenconsume CO when the CO-utilizing gene products from M. thermoacetica areexpressed.

Expression vectors were chosen from the set described by Lutz and Bujard(Lutz and Bujard, Nucleic Acids Res. 25.6:1203-1210 (1997)); these comewith compatible replicons that cover a range of copy numbers.Additionally, each contains prA1-laco1; this T7 early gene promoter isinducible by IPTG and can lead to very high levels of transcription inthe presence of IPTG and represses in other conditions. TheACS/CODH-encoding operon was cloned from Moth_(—)1204 (cooC) toMoth_(—)1197; a second version containing only Moth_(—)1203 toMoth_(—)1197 was also constructed. Both of these fragments (10-11 kbp)were confirmed by DNA sequence analysis. These were constructed in bothp15A and ColE1-based vectors for medium to high copy numbers.

To estimate the final concentrations recombinant proteins, SDS-PAGEfollowed by Western blot analyses were performed on the same cellextracts used in the CO oxidation, ACS, methyltransferase, and corrinoidFe—S assays. The antisera used were polyclonal to purified M.thermoacetica ACS/CODH and Mtr proteins and were visualized using analkaline phosphatase-linked goat-anti-rabbit secondary antibody. TheWesterns Blots are shown in FIG. 6. Amounts of CODH in ACS90 and ACS91were estimated at 50 ng by comparison to the control lanes.

A carbon monoxide oxidation assay (Seravalli et al., Biochemistry43.13:3944-3955 (2004)) was used to test whether or not functionalexpression of the CODH-encoding genes from M. thermoacetica wasachieved. Cultures of E. coli MG1655 containing either an empty vector,or the vectors expressing “Acs90” or “Acs91” were grown in TerrificBroth under anaerobic conditions (with supplements of cyanocobalamin,ferrous iron, and reducing agents) until reaching medium to high densityat which point, IPTG was added to a final concentration of 0.2 mM toinduce the promoter. After 3.5 hrs of growth at 37° C., the cells wereharvested and spun down prior to lysis with lysozyme and milddetergents. There is a benchmark figure of M. thermoacetica CODHspecific activity, 500 U at 55 C or ˜60 U at 25° C. This assay employedreduction of methyl viologen in the presence of CO. This is measured at578 nm in stoppered, anaerobic, glass cuvettes. Reactions positive forCO oxidation by CODH turned a deep violet color (see FIG. 7). About 0.5%of the cellular protein was CODH as estimated by Western blotting;therefore, the data in Table 1 are approximately 50× less than the 500U/mg activity of pure M. thermoacetica CODH. Nevertheless, thisexperiment did clearly demonstrate CO oxidation activity in recombinantE. coli with a much smaller amount in the negative controls. The smallamount of CO oxidation (CH₃ viologen reduction) seen in the negativecontrols indicates that E. coli may have a limited ability to reduce CH₃viologen.

TABLE 1 Crude extract CO Oxidation Activities ACS90 7.7 m g/ml ACS9111.8 mg/ml Mta98 9.8 mg/ml Mta99 11.2 mg/ml Extract Vol OD/ U/ml U/mgACS90 10 microliters 0.073 .0376 0.049 ACS91 10 microliters 0.096 0.4940.042 Mta99 10 microliters 0.0031 0.016 0.0014 ACS90 10 microliters0.099 0.051 0.066 Mta99 25 microliters 0.012 0.025 0.0022 ACS91 25microliters 0.215 0.443 0.037 Mta98 25 microliters 0.019 0.039 0.004ACS91 10 microliters 0.129 0.66 0.056 Averages ACS90 0.057 U/mg ACS910.045 U/mg Mta99 0.0018 U/mg

This assay is an in vitro reaction that synthesizes acetyl-CoA frommethyl-tetrahydrofolate, CO, and CoA using ACS/CODH, methyltransferase,and CFeSP (Raybuck et al., Biochemistry 27.20:7698-7702 (1988)). Byadding or leaving out each of the enzymes involved, this assay can beused for a wide range of experiments, from testing one or more purifiedenzymes or cell extracts for activity, to determining the kinetics ofthe reaction under various conditions or with limiting amounts ofsubstrate or enzyme. Samples of the reaction taken at various timepoints are quenched with 1M HCl, which liberates acetate from theacetyl-CoA end product. After purification with Dowex columns, theacetate can be analyzed by chromatography, mass spectrometry, or bymeasuring radioactivity. The exact method can be determined by thespecific substrates used in the reaction.

This assay was run in order to determine if the ACS/CODH operonexpressed in E. coli expresses the Fe—S corrinoid protein activity.Therefore, ¹⁴C-labeled methyl-THF was used as a labeled substrate tomeasure acetate synthesis by radioactivity incorporation into isolatedacetate samples. Six different conditions were tested:

-   1. Purified ACS/CODH, MeTr, and CFeSP as a positive control-   2. Purified ACS/CODH with ACS90 cell extract-   3. Purified ACS/CODH with ACS91 cell extract-   4. Purified ACS/CODH, MeTr with ACS90 cell extract-   5. Purified ACS/CODH, MeTr with ACS91 cell extract-   6. Purified ACS/CODH, MeTr with as much ACS91 cell extract as    possible (excluding the MES buffer)

The reaction was assembled in the anaerobic chamber in assay vialsfilled with CO. The total reaction volume was small compared to the vialvolume, reagents were added prior to filling with CO, a gas-tightHamilton syringe was used and the reagents were kept anaerobic. Thereaction (˜60 ul total) consisted of the cell extract (except #1), CoA,Ti(III)citrate, MES (except #6), purified ACS/CODH,14C-methyl-tetrahydrofolate, methyl-viologen, and ferredoxin.Additionally, purified MeTr was added to #1, #4-6 and purified CFeSP wasadded to #1.

The reaction was carried out in the anaerobic chamber in a sand bath at55°. The final reagent added was the ¹⁴C-methyl-tetrahydrofolate, whichstarted the reaction (t=0s). An initial sample was taken immediately,followed by samples at 30 minutes, 1 hour, and 2 hours. These timepoints are not exact, as the 6 conditions were run concurrently (sincethis experiment was primarily a qualitative one). The 15 μL samples wereadded to 15 μL of 1M HCl in scintillation vials. After counting thereaction mixtures, it was determined that the corrinoid Fe—S protein inACS90 extracts was active with total activity approaching approximately⅕ of the positive control.

Within the ACS/CODH operon is encoded an essential methyltransferaseactivity that catalyzes the transfer of CH₃ from methyl-tetrahydrofolateto the ACS complex as part of the synthesis of acetyl-CoA (i.e. this isthe step that the methyl and carbonyl paths join together). Within theoperon in M. thermoacetica, the Mtr-encoding gene is Moth_(—)1197 andcomes after the main CODH and ACS subunits. Therefore, Mtr activitywould constitute indirect evidence that the more proximal genes can beexpressed.

Mtr activity was assayed by spectroscopy. Specifically, methylatedCFeSP, with Co(III), has a small absorption peak at ˜450 nm, whilenon-methylated CFeSP, with Co(I), has a large peak at ˜390 nm. Thisspectrum is due to both the cobalt and iron-sulfur cluster chromophores.Additionally, it should be noted that the CFeSP can spontaneouslyoxidize to Co(II), which creates a broad absorption peak at ˜470 nm(Seravalli et al., Biochemistry 38.18:5728-5735 (1999)). See FIG. 8 forthe results from E. coli cells containing ACS90.

To test whether or not E. coli can grow anaerobically in the presence ofsaturating amounts of CO we made up 120 ml serum bottles with 50 ml ofTerrific Broth medium (plus NiCl₂, Fe(II) NH₄SO₄, and cyanocobalamin) inanaerobic conditions. One half of these bottles were equilibrated withnitrogen gas for 30 min. and one half was equilibrated with CO gas for30 min. An empty vector (pZA33) was used as a control and that and bothACS90 and ACS91 were tested with both N₂ and CO. All were grown for 36hrs with shaking (250 rpm) at 37 C. At the end of the 36 period,examination of the flasks showed high amounts of growth in all (FIG. 9).The bulk of the observed growth occurred overnight with a long lag ofsome (low but visible) density. Inocula sizes were ˜0.5 ml from E. colistocks.

The results are shown in Table 2. Growth reached similar levels (byvisual inspection) whether or not a strain was cultured in the presenceof CO or not. Furthermore, the negative control had a final COconcentration of 930 micromolar vs. 688 and 728 micromolar for theACS/CODH operon expressing strains. Clearly, the error in thesemeasurements is high given the large standard deviations. Nevertheless,this test does allow two tentative conclusions: 1) E. coli can tolerateexposure to CO under anaerobic conditions, and 2) E. coli cellsexpressing the ACS/CODH operon might be metabolizing some of the CO. Thesecond conclusion is significantly less certain than the first.

TABLE 2 Carbon Monoxide Concentrations, 36 hours Final CO Strain andGrowth Conditions concentration (micromolar) pZA33-CO 930 ACS90-CO 638494 734 883 ave 687 SD 164 ACS91-CO 728 812 760 611 ave 728 SD 85

Example III Enhancing the Yield of 1,3-Butanediol on Sugars withWood-Ljungdahl Pathway Enzymes

In this example, we describe a non-naturally occurring microorganismexpressing genes encoding enzymes that catalyze the carbonyl-branch ofthe Wood-Ljungdahl pathway. Wood-Ljungdahl pathway enzymes assimilatecarbon in the form of formate, CO and/or CO₂ into acetyl-CoA, which cansubsequently be converted to useful chemical products such as1,3-butanediol. The Wood-Ljungdahl pathway can also serve as a secondarycarbon assimilation pathway during growth on other substrates such asglucose. Specifically, the conversion of one mole of glucose to twomoles of acetyl-CoA generates both reducing equivalents and CO₂. The WLpathway enzymes can harness the reducing equivalents to convert the CO₂and/or formate to additional acetyl-CoA that can be further used for1,3-butanediol formation.

1,3-Butanediol can be synthesized from acetyl-CoA by several alternateroutes described previously in this application and shown in FIGS. 4 and5. The maximum achievable 1,3-butanediol yield for any of these pathwaysfrom glucose, for example, is 1 mol/mol (0.5 g/g) in the absence of theWood-Ljungdahl pathway enzymes. Additional assimilation of CO₂ viaWood-Ljungdahl pathway further improves the yield to the stoichiometrictheoretical maximum of 1.09 mol/mol (0.545 g/g). A predicted fluxdistribution for achieving the maximum theoretical yield is shown inFIG. 10.

Additionally, methanol can be co-fed with a carbohydrate such as glucoseto increase the yield of 1,3-butanediol. For example, utilizing glucoseand methanol in a 1.0:0.4 ratio affords an increase from 1 mol1,3-butanediol/mol glucose (0.5 g/g) to 1.2 mol 1,3-butanediol/molglucose. A predicted flux distribution for achieving this increasedyield is shown in FIG. 11.

Example IV Engineering Cobalamin Synthesis into an Organism

The key enzyme of the Wood-Ljungdahl pathway, ACS/CODH, requirescobalamin (vitamin B₁₂) to function. B₁₂ is synthesized de novo in someorganisms but must be supplied exogenously to others. Still otherorganisms such as S. cerevisiae lack the ability to efficiently uptakeB₁₂. This example describes a strategy for engineering de novo B₁₂synthetic capability into an organism.

B₁₂ biosynthetic pathways have been characterized in several organismsincluding Salmonella typhimurium LT2 (Roth et al., J. Bacteriol.175:3303-3316), Lactobacillus reuteri CRL1098 (Harms and Thauer, Eur. J.Biochem. 235:653-659 (1996)) and Bacillus megaterium (Brey et al., J.Bacteriol. 167:623-630 (1986)). Bacterial B₁₂ biosynthesis pathwaysinvolve 20-30 genes clustered together in one or more operons. Twocobalamin biosynthesis pathways: late-insertion (aerobic only) andearly-insertion (anaerobic) have been described (Scott, A. I., J. Org.Chem. 68:2529-2539 (2003)). The final products of the biosynthesis ofvitamin B₁₂ are 5′-deoxyadenosylcobalamin (coenzyme B₁₂) andmethylcobalamin (MeCbl). Vitamin B₁₂ is defined as cyanocobalamin(CNCbl) which is the form commonly prepared in industry. In thisexample, B₁₂ refers to all three analogous molecules.

The anaerobic cobalamin biosynthesis pathway has been well-characterizedin Salmonella typhimurium LT2 (Roth et al., J. Bacteriol.175:3303-3316)). Pathway genes are clustered in a large operon termedthe cob operon. A plasmid containing the following 20 genes from the coboperon (pAR8827) was transformed into E. coli and conferred the abilityto synthesize cobalamin de novo (Raux et al., J. Bacteriol. 178:753-767(1996)). To further improve yield of the cobyric acid precursor, theauthors removed the known regulatory elements of cbiA and altered theRBS. The genes and corresponding GenBank identifiers and gi numbers arelisted below.

cysG NP_462380.1 16766765 Salmonella typhimurium LT2 cbiK NP_460970.116765355 Salmonella typhimurium LT2 cbiL NP_460969.1 16765354 Salmonellatyphimurium LT2 cbiH NP_460972.1 16765357 Salmonella typhimurium LT2cbiF NP_460974.1 16765359 Salmonella typhimurium LT2 cbiG NP_460973.116765358 Salmonella typhimurium LT2 cbiD NP_460977.1 16765362 Salmonellatyphimurium LT2 cbiJ NP_460971.1 16765356 Salmonella typhimurium LT2cbiE NP_460976.1 16765361 Salmonella typhimurium LT2 cbiT NP_460975.116765360 Salmonella typhimurium LT2 cbiC NP_460978.1 16765363 Salmonellatyphimurium LT2 cbiA NP_460980.1 16765365 Salmonella typhimurium LT2fldA NP_459679.1 16764064 Salmonella typhimurium LT2 cobA P31570.1399274 Salmonella typhimurium LT2 cbiP AAA27268.1 154436 Salmonellatyphimurium LT2 cbiB Q05600.1 543942 Salmonella typhimurium LT2 cobUNP_460963.1 16765348 Salmonella typhimurium LT2 cobT NP_460961.116765346 Salmonella typhimurium LT2 cobS AAA27270.1 154438 Salmonellatyphimurium LT2 cobC NP_459635.1 16764020 Salmonella typhimurium LT2

Some organisms unable to synthesize B₁₂ de novo are able to catalyzesome steps of the pathway. E. coli, for example, is unable to synthesizethe corrin ring structure but encodes proteins that catalyze severalreactions in the pathway (Raux et al. J. Bacteriol. 178:753-767 (1996)).The cysG gene encodes a functional CysG, a multifunctional enzyme thatconverts uroporphyrinogen III to precorrin-2 (Hugler et al., J.Bacteriol. 184:2404-2410 (2002); Ishige et al., Appl. Environ.Microbiol. 68:1192-1195 (2002)). The proteins encoded by cobTSUtransform cobinamide to cobalamin and introduce the 5′-deoxyadenosylgroup (Raux et al., supra (1996)).

cobT NP_416495.1 16129932 Escherichia coli K12 sp. MG1655 cobSNP_416496.1 16129933 Escherichia coli K12 sp. MG1655 cobU NP_416497.116129934 Escherichia coli K12 sp. MG1655 cysG NP_417827.1 16131246Escherichia coli K12 sp. MG1655

S. cerevisiae is not able to synthesize B₁₂ de novo, nor is it able touptake the vitamin at detectable levels. However, the S. cerevisiaegenome encodes two proteins, Met1p and Met8p, that catalyze several B₁₂pathway reactions. Met1p is analogous to the uroporphyrinogen IIItransmethylase CysG of S. typhimurium, which catalyzes the first step ofB 12 biosynthesis from uroporphyrinogen III (Raux et al., Biochem. J.338(pt. 3):701-708 (1999)). The Met8p protein is a bifunctional proteinwith uroporphyrinogen III transmethylase activity and cobaltochelataseactivity analogous to the CysG of B. megaterium (Raux et al., supra(1999)).

Met1p NP_012995.1 6322922 Saccharomyces cerevisiae Met8p NP_009772.16319690 Saccharomyces cerevisiae

Any or all of these genes can be introduced into an organism deficientin one or more components of cobalamin synthesis to enable or increasethe efficiency of cobalamin synthesis.

Example V Engineering Enhanced Cobalamin Uptake Capability in anOrganism

This example describes engineering B₁₂ uptake capability into a hostorganism. B₁₂ uptake requires a specific transport system (Sennett etal., Annu. Rev. Biochem. 50:1053-1086 (1981)). The B₁₂ transport systemof E. coli has been extensively studied. High-affinity transport acrossthe outer membrane is calcium-dependent and mediated by a 66 kDa outermembrane porin, BtuB (Heller et al., J. Bacteriol. 161:896-903 (1985)).BtuB interacts with the TonB energy transducing system (TonB-ExbB-ExbD),facilitating energy-dependent translocation and binding to periplasmicbinding protein BtuF (WO/2007/141208; Atsumi et al., Nature 451:86-89(2008)). Transport across the inner membrane is facilitated by an ABCtype uptake system composed of BtuF, BtuD (ATP binding component) andBtuC (permease) (Binstock et al., Meth. Enzymol. 71(pt. C):403-411(1981)). Crystal structures of the BtuCDF complex are available (Atsumiet al., supra (2008); Binstock et al., supra (1981)). An additionalprotein, BtuE, is coexpressed in the btuCED operon, but this protein isnot required for B12 transport and its function is unknown (Rioux etal., Mol. Gen. Genet. 217:301-308 (1989)). The btuCED operon isconstitutively expressed. The GenBank identifiers and GI numbers of thegenes associated with B₁₂ transport are listed below.

btuB NP_418401.1 16131804 Escherichia coli K12 sp. MG1655 btuCNP_416226.1 16129667 Escherichia coli K12 sp. MG1655 btuD NP_416224.116129665 Escherichia coli K12 sp. MG1655 btuF NP_414700.1 16128151Escherichia coli K12 sp. MG1655 tonB NP_415768.1 16129213 Escherichiacoli K12 sp. MG1655 exbB NP_417479.1 16130904 Escherichia coli K12 sp.MG1655 exbD NP_417478.1 16130903 Escherichia coli K12 sp. MG1655

The B₁₂ uptake capability of an organism can be further improved byoverexpressing genes encoding the requisite transport proteins, andreducing or eliminating negative regulatory control. Overexpressing thebtuBCDF genes leads to increased binding of B12 to membranes andincreased rate of uptake into cells. Another strategy is to removeregulatory control. The btuB mRNA translation is directly repressed byB12 at the 5′ UTR (Nahvi et al., Chem. Biol. 9:1043 (2002)). Thisinteraction may induce mRNA folding to block ribosome access to thetranslational start. Mutation or elimination of the B₁₂ binding siteremoves inhibition and improves the efficiency of B₁₂ uptake (U.S. Pat.No. 6,432,686, Bulthuis et al.). These strategies were successfullyemployed to improve B₁₂ uptake capability in 1,3-PDO producingmicroorganisms (WO/1999/058686) and (U.S. Pat. No. 6,432,686, Bulthuiset al.). A recent patent application describes improving the efficiencyof B₁₂ uptake (WO/2008/152016) by deleting negative regulatory proteinssuch as C. glutamicum btuR2.

S. typhimurium possesses both high and low affinity transporters forB₁₂. The high affinity transporter is encoded by btuB (Rioux et al., J.Bacteriol. 171:2986-2993 (1989)). Like E. coli transport across theperiplasmic membrane is predicted to occur via an ABC transport system,although this has not been characterized to date. The B₁₂ bindingprotein is encoded by btuD and btuE, and btuC is predicted to encode thepermease.

btuB AAA27031.1 153891 Salmonella typhimurium LT2 btuC NP_460306.116764691 Salmonella typhimurium LT2 btuD NP_460308.1 16764693 Salmonellatyphimurium LT2 btuE AAL20266.1 16419860 Salmonella typhimurium LT2

Any or all of these genes can be introduced into an organism deficientin one or more components of cobalamin uptake to enable or increase theefficiency cobalamin uptake.

Method for quantifying B₁₂ in the culture medium. To quantify the amountof B₁₂ in the culture medium, cell free samples are run on HPLC.Cobalamin quantification is achieved by comparing peak area ratios at278 nm and 361 num with standards, then applying peak areas to standardcurves of cobalamin.

Throughout this application various publications have been referencedwithin parentheses. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific examples and studies detailed above are onlyillustrative of the invention. It should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1. A non-naturally occurring microbial organism, having a 1,3-butanediol(1,3-BDO) pathway, wherein said microbial organism comprises anexogenous nucleic acid encoding each of the following a 1,3-BDO pathwayenzymes or proteins expressed in a sufficient amount to produce1,3-BDO: 1) formate dehydrogenase that catalyzes the incorporation ofCO2 into formate; 2) formyltetrahydrofolate synthetase that ligatesformate to tetrahydrofolate to form 10-formyltetrahydro folate; 3)methenyltetrahydrofolate cyclohydrolase that converts10-formyltetrahydrofolate to methyltetrahydrofolate; 4)methylenetetrahydrofolate dehydrogenase that convertsmethyltetrahydrofolate to methenyltetrahydrofolate; 5)methylenetetrahydrofolate reductase that convertsmetheneyltetrahydrofolate to 5-methyltetrahydrofolate; 6)methyltetrahydrofolate:corrinoid protein methyltransferase thatcatalyzes the transfer of a methyl group from 5-methyltetrahydrofolateto corrinoid iron sulfur protein; 7) corrinoid iron-sulfur protein; 8)nickel-protein assembly protein; 9) ferredoxin; 10) acetyl-CoA synthasethat catalyzes the condensation of the methylated corrinoid iron sulfurprotein, carbon monoxide and coenzyme A, yielding acetyl-CoA; 11) carbonmonoxide dehydrogenase that converts CO and water to CO2 while passingthe electrons to a reduced acceptor; 12) hydrogenase that transferselectrons from H2 to an acceptor; 13) acetoacetyl-CoA thiolase thatconverts acetyl-CoA to acetoacetyl-CoA; 14) acetoacetyl-CoA reductase(ketone reducing) that converts acetoacetyl-CoA to 3-hydroxybutyryl-CoA;15) 3-hydroxybutyryl-CoA reductase (aldehyde forming) that converts3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde; and 16)3-hydroxybutyraldehyde reductase that converts 3-hydroxybutyraldehyde to1,3-BDO.
 2. The non-naturally occurring microbial organism of claim 1,wherein said microbial organism uses a carbon feedstock selected from 1)CO, 2) CO₂ and H₂, 3) CO, CO₂, and H₂, 4) synthesis gas comprising COand H₂, 5) synthesis gas comprising CO, CO₂, and H₂, and 6) one or morecarbohydrates.
 3. The non-naturally occurring microbial organism of 2,wherein the carbon feedstock is CO.
 4. The non-naturally occurringmicrobial organism of 2, wherein the carbon feedstock is CO₂ and H₂. 5.The non-naturally occurring microbial organism of 2, wherein the carbonfeedstock is CO, CO₂, and H₂.
 6. The non-naturally occurring microbialorganism of 2, wherein the carbon feedstock is synthesis gas comprisingCO and H₂.
 7. The non-naturally occurring microbial organism of 2,wherein the carbon feedstock is synthesis gas comprising CO, CO₂, andH₂.
 8. The non-naturally occurring microbial organism of 2, wherein thecarbon feedstock is one or more carbohydrates.